Construction and application of T7-like expression system in Escherichia coli Nissle 1917

ZHENG Ye, DENG Ruizhe, YANG Furen, LIN Yina, WANG Hui, YE Jianwen

Journal of Jinan University Natural Science & Medicine Edition ›› 2025, Vol. 46 ›› Issue (2) : 151-162.

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Journal of Jinan University Natural Science & Medicine Edition ›› 2025, Vol. 46 ›› Issue (2) : 151-162. DOI: 10.11778/j.jdxb.20250056
Research on basic medicine: Biomedical materials

Construction and application of T7-like expression system in Escherichia coli Nissle 1917

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Abstract

Objective: To construct a strong inducible expression system based on T7-like RNA polymerase MmP1 in Escherichia coli Nissle 1917 (EcN) to achieve efficient expression of the target protein. Methods: Constructing different inducible expression systems in EcN for “dose-response” characterization to screen out the best-performing induced system with low leakage and high induction expression level. Optimizing the expression output and dynamic range of the T7-like system by integrating the T7-like RNA polymerase (MmP1) expression on the chromosome of EcN, namely strain LM01, thereby constructing promoter mutation library of PMmP1 for tunable induced expression output. T7-like induced system based on MmP1 was used to control the expression of superoxide dismutase (SOD) of high yield, giving strong evidence for protein expression of T7-like system in EcN. Results: Several inducible systems were successfully constructed and characterized in EcN. Of which, the T7-like induction system based on MmP1 RNA polymerase displayed the best-performing saturated induction output with maximum dynamic range reaching up to 909-fold. After the MmP1 polymerase expression module was integrated into the genome of EcN, the saturated induction level was increased by 66.8%. The construction of PMmP1 promoter library provides a wide range of inducible expression output (dynamic range), spanning 65 to 1 097 fold. The T7-like (MmP1) system demonstrated the highest soluble SOD protein production yield of 435.7 mg/L, which was 4.02-fold higher than vanillic acid induction system. In addition, the maximum enzymatic activity of SOD achieves 391.3 U/mL. Conclusion: T7-like (MmP1) induction system can be successfully constructed in EcN with high saturated expression level, low basal leakiness, as well as controllable dynamic range of tunable gene expression control for efficient protein synthesis in the coming future.

Key words

Escherichia coli Nissle 1917 / inducible expression system / T7-like expression system / protein expression / synthetic biology

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ZHENG Ye , DENG Ruizhe , YANG Furen , LIN Yina , WANG Hui , YE Jianwen. Construction and application of T7-like expression system in Escherichia coli Nissle 1917. Journal of Jinan University Natural Science & Medicine Edition. 2025, 46(2): 151-162 https://doi.org/10.11778/j.jdxb.20250056
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大肠杆菌Nissle 1917(Escherichia coli Nissle 1917,EcN),因其无内毒素及具备益生特性等优势,已逐渐成为药物分子合成及活体治疗递送系统等医学应用领域极具潜力的合成生物学底盘之一[1-2]。一方面,工程化改造的活体EcN已被广泛应用于肠道炎症[3-5]、肿瘤[6-8]及代谢性疾病[9-10]等治疗研究领域,其治疗功能的有效发挥主要基于其治疗性蛋白质(如酶、细胞因子)及小分子代谢物的合成与递送。如通过工程化EcN表达超氧化物歧化酶(superoxide dismutase,SOD)可清除活性氧分子,从而缓解炎症中的氧化应激损伤[4-5,11]。另一方面,工程化EcN可实现具有治疗性功能的高附加值蛋白的生物合成,这展示出一定的产业化前景。因此,为充分发挥该微生物底盘的应用潜力,开发与其细胞代谢适配的蛋白高效表达系统具有重要的意义。
在代谢工程中,基因表达调控主要有静态调控策略及动态调控策略。其中,静态调控策略是当前EcN中较为基础和核心的表达调控方式。通过基因插入或启动子元件优化[3,12]等方式,该策略能实现目标基因的定向有效表达。然而,不断变化的代谢环境及持续累积的目标蛋白可能会使工程菌的生长能力受到抑制,不利于产量的提升[13]。同时,在复杂的体内应用环境下,目标蛋白的异位表达和释放会存在潜在的安全风险。因此,基于生物传感设计的动态调控策略一定程度上更契合工程化EcN的应用需求。利用该策略,工程菌可以实现报告蛋白及治疗分子的定向释放,可用于疾病的诊断和治疗[14]。此外,动态调控系统可用于最适基因表达强度的探索,对静态调控策略的改进具有重要的指导意义。然而,相较其他模式大肠杆菌,现有报道的EcN动态调控表达系统种类仍相对有限[1,15 -17],在饱和输出强度、动态调控范围等参数上仍存在较大的局限。
为了在EcN中实现蛋白的可控诱导表达,不同的基因表达调控系统均有着潜在的解决能力:首先,各种以配体响应为基础的诱导调控系统[18-19],其通过转录调节因子与小分子诱导剂相互作用,进而与相应的启动子操纵子区域结合,以此实现正交性地精准调控蛋白表达;其次,基于T7或类T7的RNA聚合酶(RNA polymerase,RNAP)的基因强表达系统[20-22],凭借高转录活性,已在大肠杆菌BL21(DE3)中建立了成熟的蛋白表达平台。而且,Morganella噬菌体来源的RNAP MmP1被开发为盐单胞菌TD01的新型类T7诱导表达系统[23-24],其具备T7系统高效转录及严格调控特性的同时,还展现出跨物种(如大肠杆菌S17-1、嗜昆虫假单胞菌LAC31等)适配优势。然而,在非模式菌株中进行调控元件移植时,由于自身代谢及调控的复杂性,可能出现表达元件不匹配、宿主-元件干扰等情况,从而导致其响应调控功能下降或失效,因此需要进一步开展适配性改造等研究[23,25]
为应对上述挑战,本研究尝试在EcN中系统地构建并表征不同类型的诱导调控系统。目前在EcN首次构建了性能优异的类T7(MmP1)诱导表达系统,并实现治疗性蛋白的表达测试及优化。旨在构建响应输出高、本底渗漏低、动态范围可调的基因表达调控系统,从而扩展EcN的合成生物学的工具箱,为该底盘在医学治疗及代谢工程领域的广泛应用提供有效可用的调控策略。

1 材料与方法

1.1 材料

1.1.1 菌株与质粒

采用的菌株为大肠杆菌,其中EcN作为表达宿主,大肠杆菌S17-1用于质粒构建。使用表达质粒主要基于pSEVA321载体骨架进行构建。使用的菌株及质粒如表12所示,部分合成引物序列如表3所示。
Table 1 Strains used in this study

表1 实验使用菌株

菌株名称 描述 来源
EcN 大肠杆菌Nissle 1917的野生型菌株 实验室保存
E. coli S17-1 用于重组质粒的构建 实验室保存
EcN LM01 EcN的衍生菌株,在基因组整合了lacI-RNAPMmP1模块 本研究
Table 2 Plasmids used in this study

表2 实验使用质粒

质粒名称 描述 来源
pSEVA321-araC-araE-PBAD-sfgfp pSEVA321载体,PBAD启动子控制sfgfp表达 文献[18]
pSEVA321-LuxR-PLux-sfgfp pSEVA321载体,PLux启动子控制sfgfp表达 文献[18]
pSEVA321-lacI-Ptac-sfgfp pSEVA321载体,Ptac启动子控制sfgfp表达 文献[18]
pSEVA321-vanR-PVan-sfgfp pSEVA321载体,PVan启动子控制sfgfp表达 文献[18]
pSEVA321-lacI-RNAPMmP1-PMmP1-sfgfp pSEVA321载体,包含lacI-RNAPMmP1模块,PMmP1启动子控制sfgfp表达 本研究
pSEVA321-lacI-RNAPT7-PT7-sfgfp pSEVA321载体,包含lacI-RNAPT7模块,PT7启动子控制sfgfp表达 本研究
pSEVA321-PMmP1-sfgfp pSEVA321载体,PMmP1启动子控制sfgfp表达 文献[23]
pSEVA321-PMmP1 library-sfgfp pSEVA321-PMmP1-sfgfp衍生,PMmP1部分序列进行了定点突变 文献[23]
pSEVA321-PMmP1-sod pSEVA321-PMmP1-sfgfp衍生,用sod替换sfgfp 本研究
pSEVA321-vanR-PVan-sod pSEVA321-vanR-PVan-sfgfp衍生,用sod替换sfgfp 本研究
p46cas9 表达λ-Red和SpCas9,用于EcN的遗传改造 文献[26]
Ts-PG-LM 供体质粒,用于EcN的遗传改造 本研究
Table 3 Primers used in this study

表3 实验使用引物

引物名称 引物序列(5'→3')
321B-F TCTAGAGCTCGGTACCAAATTCCA
321B-R TCCTGTGTGAAATTGTTATCCGCT
M-F CAATTTCACACAGGAAAATTAATATCTGAAAGAATCATAGGCTTGGA
I-R GTACCGAGCTCTAGAGCAGCCTGCCAGATTCT
T-F CAATTTCACACAGGATTACGCGAACGCGAAGTC
S-F GAGGAGAAATACTAGATGTCATTCGAATTACCTGCACT
S-R GGAGTGACGATTATGCAGCGAGATTTTTCGCTACG
g1-F TTGTCTGGAAGCGGCGATGGACTAGTATTATACCTAGGACTGAGCTAGCT
g2-R CCATCGCCGCTTCCAGACAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCT
R-F CTAGTATTTCTCCTCTTTCTCTAGTATTAAACAAAA
R-R TCGTCACTCCACCGGTGCTTAATAA

1.1.2 培养基与诱导剂

采用溶菌肉汤(Luria-Bertani,LB)培养基作为大肠杆菌的基础培养及表达体系,其配方为氯化钠10 g/L、胰蛋白胨10 g/L、酵母粉5 g/L。固体LB培养基在液体LB培养基中额外添加1.9 g/L的琼脂进行制备。根据实验需求,在培养基制备过程中添加25 μg/mL氯霉素或50 μg/mL卡那霉素。诱导剂配制方式如下:异丙基-β-D-硫代半乳糖苷(isopropyl β-D-thiogalactopyranoside,IPTG)(BBI,中国)、L-阿拉伯糖(L-arabinose,L-Ara)(BBI,中国)、脂肪酰基高丝氨酸内酯(3-oxo-hexanoyl-homoserine lactone,OHC6)(Sigma-Aldrich,美国)和脱水四环素(anhydrotetracycline,aTc)(源叶生物,中国)使用ddH2O溶解,香草酸(vanillic acid,Van)(Macklin,中国)使用二甲基亚砜(dimethyl sulfoxide,DMSO)(Macklin,中国)溶解。所有诱导剂根据实验需求配置不同浓度梯度,并使用0.22 μm孔径的水相或有机相针式过滤器(天津津腾,中国)进行过滤除菌。

1.2 实验方法

1.2.1 大肠杆菌感受态细胞的制备

使用固体LB培养基平板进行大肠杆菌的活化,将活化后的单克隆菌落接种于LB培养基,在37 ℃、220 r/min条件下过夜培养16 h。随后按体积分数1%将菌液接种至50 mL液体LB培养基,在37 ℃、220 r/min条件下培养至菌液光密度D(600 nm)达到0.3~0.5,立即于4 ℃、6 000 r/min条件下离心5 min收集菌体,用5 mL预冷的液体LB培养基重悬菌体,并加入等体积的一步法感受态制备试剂(Boyetime,中国),轻柔混匀后冰浴10 min,随后分装并于-80 ℃保存备用。此外,在制备含有p46Cas9质粒的大肠杆菌感受态细胞时,需在30 ℃条件下进行培养,并在培养基中添加12.5 μg/mL氯霉素以防止质粒丢失。

1.2.2 质粒构建及转化

采用Gibson Assembly方法[27]构建质粒。所用引物通过Snapgene 6.0.2软件进行设计与优化,并使用NEB Tm Calculator(https://tmcalculator.neb.com)计算引物对应的退火温度。DNA模板来源于实验室保存菌株或由上海生工生物工程有限公司合成。在聚合酶链式反应(polymerase chain reaction,PCR)扩增目标片段后,所有线性DNA片段通过凝胶电泳分离并提取纯化,然后使用无缝克隆试剂盒(Genstar,中国)进行组装连接。将反应混合物加入感受态细胞后进行化学转化,并涂布于含有相应抗生素的固体LB平板上,过夜培养后挑选单克隆进行菌落PCR及测序验证。实验涉及的测序分析由上海生工生物工程有限公司提供技术支持。

1.2.3 EcN的遗传改造

使用λ-Red及CRISPR/Cas9技术进行目标片段的基因组整合[26]。首先,将p46cas9辅助质粒和Ts-PG系列供体质粒通过化学转化导入EcN中,阳性转化子在30 ℃、220 r/min条件下过夜培养。随后,向培养体系中添加终浓度为5 mg/mL的L-Ara和80 ng/mL的aTc继续培养12 h。将菌液用LB培养基进行稀释200倍,随后涂布于含有12.5 μg/mL氯霉素、50 μg/mL卡那霉素及2 g/L L-Ara的LB固体培养基平板上。于30 ℃条件下过夜培养后,采用菌落PCR方法对转化子进行基因型鉴定,并且进行DNA测序,验证目标片段是否成功插入。整合成功的单克隆接种至不含抗生素的LB培养基,在42 ℃条件下培养进行质粒消除。

1.2.4 荧光强度检测

将携带sfgfp报告基因表达质粒的大肠杆菌活化后接种至LB培养基,在37 ℃培养12 h作为种子液。随后,在96孔深孔板的各孔中加入1 mL LB培养基,按照实验需求分别添加不同种类及浓度的诱导剂并进行充分混合,按体积分数1%将种子液接种至对应体系中,在恒温振荡器中培养24 h,每个实验组设置3个生物学重复。培养结束后,取适量菌液,在4 ℃、12 000 r/min条件下离心2 min以收集菌体,弃上清液,随后使用PBS重悬菌体,并按照相同倍数进行稀释,保证各组样品D(600 nm)均介于0.2至0.8。取200 μL菌体悬液至96孔酶标板中,使用Varioskan Flash多功能酶标仪(ThermoFisher Scientific,美国)进行荧光强度(fluorescence intensity,FI)和细胞密度[D(600 nm)]测定,各参数设置为:荧光激发波长488 nm, 荧光发射波长520 nm, 光密度波长600 nm。最终根据D(600 nm)对荧光强度进行归一化计算相对荧光强度。

1.2.5 SOD蛋白的摇瓶发酵

将携带SOD蛋白表达质粒的大肠杆菌经平板划线活化后接种至LB培养基,在37 ℃培养12 h作为种子液。按体积分数5%将种子液接种至含20 mL LB培养基的150 mL摇瓶中,在37 ℃培养1~2 h至D(600 nm)达0.8~1.0,随后添加IPTG或Van进行蛋白的诱导表达,在37 ℃、220 r/min条件下发酵20 h。

1.2.6 蛋白表达量的测定

深孔板培养或摇瓶发酵结束后,取菌液在4 ℃、6 000 r/min条件下离心10 min以收集菌体,弃上清液,随后使用PBS重悬菌体并进行超声破碎。所得裂解液在4 ℃、12 000 r/min条件下离心10 min,尽可能收集上清液,弃去不可溶的细胞碎片沉淀。使用BCA蛋白质试剂盒(Boyetime,中国)测定上清液中可溶性蛋白质的总含量。进一步使用SDS-PAGE电泳分析,经考马斯亮蓝染色法染色后,使用平板扫描仪(EPSON,日本)扫描成像。根据凝胶上各蛋白条带的灰度值,使用Quantity One 4.6.2(Bio-Rad,美国)计算可溶性蛋白质中目标蛋白的占比,用于测定目标蛋白的含量。

1.2.7 SOD蛋白的活性检测

本研究采用WST-8法对破碎上清液中的可溶性SOD进行活性检测。使用WST-8法总SOD活性检测试剂盒(Boyetime,中国)进行,样品按实际情况进行稀释以保证抑制百分率在30%~70%,根据450 nm吸光值及稀释倍数计算酶活力。

2 结果

2.1 不同诱导表达系统在EcN中的构建及表征

本研究在EcN中系统构建了两类诱导表达系统,分别是基于配体分子结合调控蛋白响应调控的诱导表达系统和基于噬菌体来源的T7或类T7 RNA聚合酶(MmP1)的强诱导表达系统。不同诱导系统的核心调控元件均构建在以RK2为复制子的pSEVA321载体质粒上,包括配体分子结合的调控蛋白编码基因(araC-L-Ara、luxR-OHC6、lacI-IPTG、vanR-Van)和IPTG诱导表达用于级联调控的T7及类T7 RNA聚合酶编码基因、受调控复合体的启动子、编码绿色荧光蛋白(sfGFP)的报告基因(图1A)。随后,将这些质粒分别转化至EcN中,并对不同诱导调控系统在零诱导和饱和诱导条件下的24 h绿色荧光蛋白表达水平进行相对荧光强度表征及SDS-PAGE电泳分析(图1B)。结果表明,相较其他诱导系统,由类T7 RNAP驱动的MmP1诱导表达系统在EcN中表现出最优性能,饱和诱导输出强度最高,sfGFP表达量达314.1 mg/L。同时,该系统本底渗漏最低,动态调控范围最大,达到909倍。其次,Van响应的vanR-PVan系统在不同配体响应的诱导表达系统中表现最佳,其饱和诱导输出水平较高,且动态范围达到42.9倍。相比之下,常规的T7系统在零诱导状态下表现出显著的本底渗漏,而诱导后输出水平反而降低至零诱导时的22%,这一结果与其在BL21(DE3)中的表现相悖。随后,通过SDS-PAGE电泳对各诱导系统的sfgfp表达情况开展了进一步验证(图1C),结果所呈现的趋势与上述荧光表征情况一致。因此,本研究初步选定类T7 MmP1系统在EcN中开展进一步的优化及应用。
Figure 1 Construction and characterization of different inducible systems in EcN
(A)Schematic of the inducible systems engineered in EcN. Left: Conventional inducible systems based on ligand-responsive promoters. Right: T7 expression system and T7-like MmP1 expression system. The systems were constructed on the pSEVA321 vector, respectively; (B)Characterization of expression levels driven by the above inducible systems in EcN. Inducer concentrations uesd: L-Ara (10-3 mol/L), OHC6 (10-5 mol/L), IPTG (10-3 mol/L), and Van (10-3 mol/L); (C)SDS-PAGE analysis of sfGFP expression under different induction systems.

图1 EcN中不同诱导调控系统的构建及表征

(A)EcN中不同诱导系统的构建示意图。左侧为基于配体响应的常规正交诱导系统,右侧为T7表达系统及类T7 MmP1表达系统,以上系统分别构建在pSEVA321骨架上;(B)不同诱导系统在EcN中的表达强度表征,其中,不同诱导剂的饱和诱导浓度使用如下:L-Ara(10-3 mol/L),OHC6(10-5 mol/L),IPTG(10-3 mol/L),Van(10-3 mol/L);(C)不同诱导系统调控下sfGFP蛋白表达的SDS-PAGE电泳结果。

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2.2 类T7 MmP1系统的遗传稳定性优化及剂量响应表征

在上述研究中,MmP1系统已在质粒上进行了初步的测试。为降低质粒产生的代谢负荷并提升调控模块的遗传稳定性,采用λ-Red和CRISPR-Cas9技术将pSEVA321-lacI-RNAPMmP1-PMmP1-sfgfp质粒中携带的lacI-RNAPMmP1调控模块整合至EcN基因组的Lon蛋白酶基因座,并成功构建LM01工程菌株(图2A)。随后,将保留PMmP1-sfgfp报告模块的pSEVA321载体质粒转化至LM01后,对其在零诱导和饱和诱导条件下的表达水平进行相对荧光强度表征(图2B)。从图中可得知,与原质粒表达体系相比,基因组整合菌中PMmP1启动子在10-3 mol/L IPTG诱导条件下的饱和诱导输出强度提高66.8%,且动态调控范围提升到1 097倍,跨越3个数量级。随后,对该诱导系统从0.5×10-7到10-3 mol/L IPTG的剂量-响应曲线进行表征(图2C),以进一步细化评估其在EcN中的诱导表达调控性能。结果表明,该系统的IPTG浓度响应范围为(10-7~0.5×10-4) mol/L。在0.5×10-4 mol/L的IPTG浓度(低于常用的1×10-3 mol/L)诱导下,该系统接近实现饱和诱导输出,荧光表达强度达到了最高值的96.6%。
Figure 2 Robustness improvement and characterization of the T7-like inducible system in EcN
(A)Schematic for integrating the RNAPMmP1 module into the EcN genome. The lacI-RNAPMmP1 module was integrated into the genome, while the PMmP1 promoter expression module was constructed on the pSEVA321 vector; (B)Inducible expression levels of the PMmP1 promoter of above modules. ****P<0.000 1; (C)Dose-response functions of the MmP1 system in strain LM01 under varying IPTG concentrations.

图2 EcN中类T7 MmP1系统的遗传稳定性优化及表征

(A)RNAPMmP1模块整合EcN基因组流程。将lacI-RNAPMmP1模块整合至EcN的基因组,PMmP1启动子表达模块构建在pSEVA321载体上;(B)PMmP1启动子在上述模块构建方式下的诱导响应表达水平表征,****P<0.000 1;(C)LM01中MmP1系统对IPTG的剂量-响应曲线。

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2.3 PMmP1启动子文库测试与动态调控范围扩展

动态调控范围反映了启动子在零诱导和饱和诱导条件下的输出差异,在实际应用中至关重要。为进一步扩展类T7 MmP1诱导表达系统的应用潜力,针对MmP1系统另一核心元件PMmP1启动子区域展开了优化和探索。该启动子的-12区至-7区,该区是与RNAP结合的核心区域,对该系统的诱导表达稳定性有着重要的影响作用(图3A)。因此,选择该区域外-1区至-4区进行随机突变以建立启动子文库进行测试。启动子变体在LM01中的零诱导和饱和诱导输出的相对荧光强度表征结果表明,多种PMmP1启动子变体的饱和诱导输出水平呈现显著的强弱变化,且动态调控范围广,涵盖了1 097倍(WT)、163倍(TCTT)、129倍(TAGG)及65倍(CCTT)等高中低多个跨度(图3B)。随后,对不同强度的PMmP1启动子变体进行IPTG诱导剂的剂量-响应曲线表征测试(图3C)。结果表明,3种变体的IPTG响应范围分别为(10-7~0.5×10-3) mol/L(TCTT)、(0.5×10-6~0.5×10-3) mol/L(TAGG)、(10-7~10-4) mol/L(CCTT)。综上,WT具有最宽广的动态调控范围,变体(TCTT)具有最大的IPTG响应范围。
Figure 3 Constructing PMmP1 promoter library to achieve broad and precise gene expression control
(A)Schematic of constructing the PMmP1 promoter library; (B)Inducible expression levels of PMmP1 promoter mutants in LM01; (C)Dose-response functions of promoter mutants under varying IPTG concentrations.

图3 构建PMmP1启动子文库以实现不同表达调控水平的精确调控

(A)PMmP1启动子变体库构建示意图;(B)PMmP1启动子变体LM01中的诱导响应表达水平表征;(C)不同动态范围启动子变体对IPTG的剂量-响应曲线。

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2.4 类T7 MmP1系统在治疗性蛋白表达中的应用验证

为评估MmP1系统在蛋白表达中的实际能力,本研究选取SOD(24.2 kD)作为治疗性蛋白的代表性案例进行表达测试。为实现SOD蛋白高效表达,以文库中饱和输出水平最高的野生型PMmP1启动子为基础进行SOD表达质粒的构建。同时,选取常规诱导系统中响应输出水平相对较高的Van诱导系统作为其对照(图4 A)。各组工程菌在不同诱导条件下进行摇瓶发酵,后续通过SDS-PAGE电泳及酶活力检测对发酵产物进行评估。结果显示,Van诱导系统在饱和诱导下可溶性SOD表达量达到108.5 mg/L;MmP1诱导表达系统则在10-3 mol/L IPTG诱导下可溶性SOD表达产量达到了435.7 mg/L,约为Van诱导系统最高产量的4.02倍(图4B、C)。同时,在10-4 mol/L IPTG诱导条件下,工程菌合成的SOD达到最高的酶活性,为391.3 U/mL(图4D)。
Figure 4 Therapeutic protein production of soluble SOD by engineered EcN using T7-like MmP1 system
(A)Schematic of SOD expression modules regulated by the T7-like MmP1 system(upper) and vanillic acid-inducible(lower) control; (B)SDS-PAGE analysis of soluble SOD expression under different induction systems; (C)Characterization of SOD protein production under above inducible systems; (D)Enzymatic activity assessment of SOD protein expressed by different inducible systems. ****P<0.000 1. Inducer concentration unit: mol/L

图4 类T7 MmP1系统应用于治疗蛋白SOD的可溶性表达

(A)类T7 MmP1系统(上)及Van诱导系统(下)的SOD表达模块构建示意图;(B)不同诱导系统调控下可溶性SOD表达的SDS-PAGE电泳结果;(C)不同诱导系统调控下表达可溶性SOD蛋白的产量及工程菌生物量;(D)不同诱导系统调控下表达可溶性SOD的酶活性。****P<0.000 1。诱导剂浓度单位:mol/L

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3 讨论

为满足基于工程化EcN活体递送疗法、蛋白工业化生产等日益增长的需求,本研究通过在EcN中构建并优化诱导表达系统,成功为该生物底盘的重组蛋白表达提供了具有潜力的调控策略。本研究首先尝试在EcN中分别构建和表征了不同的诱导调控表达系统。结果显示,各类配体响应的诱导系统在EcN中均成功构建了基因诱导表达调控回路,在诱导剂添加的条件下能实现从4.9至42.9倍的动态输出范围。尽管IPTG响应回路的功能完整性在EcN中已被验证,但是常规T7 RNAP系统在EcN中仍表现出与大肠杆菌BL21(DE3)显著不同的特性,具体表现为本底渗漏水平升高及诱导后表达水平降低(图1B)。这种差异可能源于此系统在EcN中的构建方式及相对空间位置所导致的RNAP表达强度变化[28],或者宿主干扰导致相关元件活性出现异常[23]。需通过进一步机制探索及系统优化才能实现其在EcN中的应用。与其不同的是,类T7 MmP1系统在EcN中表现出良好的适配性,其具备极高的饱和响应强度及宽广的动态调控范围,相较上述其他的诱导系统,以及现有研究中报道的J23系列组成型启动子文库都显示出一定的优势[12,17],展现出较大的应用潜力。
在后续RNAPMmP1模块基因组整合的过程中,发现PMmP1启动子的响应表达水平得到提升,这一现象可能与模块在染色体与质粒之间的表达水平差异有关。该差异受多种潜在因素影响,如整合后模块的拷贝数变化、调控模块的表达稳定性及整合位点的空间效应等[17]。此外,在PMmP1启动子文库表征过程中,不同变体除了饱和输出强度改变外,在IPTG的响应范围及饱和响应浓度上也存在差异。既往研究已确定类T7 RNAP结合核心区域位于启动子的-7至-12区,而对-1至-4区进行定向突变主要影响转录起始强度[29],但上述现象表明RNAP与启动子之间的结合效应仍然受到潜在因素的细微影响。因此,后续可针对该现象开展更深入的机制探索。从另一角度考虑,该现象也为该系统在不同应用场景下的灵活适配性改造提供了新的可能性。此外,在SOD蛋白的表达测试过程中,最高产量与最高酶活性对应的IPTG诱导浓度并不一致,可见蛋白合成过程中转录后的加工对蛋白功能的有效发挥同样具有重要意义。
MmP1系统在蛋白表达测试中展现出高效性能,但仍存在优化空间。当前该系统的激活依赖于IPTG的诱导,这不可避免地提升了工业生产成本,使其作为工业底盘的应用有所限制。再者,高浓度的IPTG也具有潜在的细胞毒性,从而会抑制工程菌的生长,限制进一步产量提升,也可能对下游治疗及产品的安全性产生不利的影响。针对上述问题,后续可根据实际应用场景对该系统中负责级联调控RNAP的元件进行调整。例如,在肠道环境中应用时,可采用脂肪酸响应的诱导系统[30](如FadR系统)对类T7 RNAP进行级联调控,增强生物相容性;再者,也可以采用温度[31-33]、氧浓度[34]、光感[35]等非化学诱导响应系统,或是内源性代谢物响应元件(如群体感应系统)实现不依赖诱导剂的级联调控[36]
此外,对于结构复杂的治疗性蛋白(如抗体[37]、干扰素[38]等)的合成,高强度的转录水平也有可能造成蛋白折叠错误或聚集,最终形成包涵体致使目标蛋白的可溶性降低,不利于其功能的正常发挥[39]。因此,可考虑增加伴侣蛋白共表达模块(如ptF16、GroEL/GroES)以提升目标蛋白的折叠效率[37-38,40],促使该系统在蛋白表达的其他环节中更加完善。这些改进有助于推动MmP1系统回路不断向模块化、多功能方向发展,形成更为完整的蛋白表达体系,从而加速EcN生物底盘从基础研究向临床治疗及工业生物制造等实际应用场景的转化进程。
综上所述,本研究在EcN中系统地构建和测试了多种诱导调控系统,其中首次构建了具有高响应强度、低本底渗漏、动态范围可调的类T7 MmP1诱导表达调控系统,实现了治疗性蛋白SOD的可溶性高效合成。该系统不仅丰富了EcN的合成生物学工具箱,更为开发基于 EcN 的活体递送疗法及蛋白合成产业化生产提供了新的策略。

作者贡献声明

郑烨:实验设计及实验操作、图表绘制、撰写论文;邓睿哲、杨富荏:实验操作;林艺娜:论文框架、撰写指导;王辉:思路拟定,撰写指导;叶健文:思路拟定,指导性支持,撰写指导。

利益冲突声明

本研究未受到企业、公司等第三方资助,不存在潜在利益冲突。

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MEYER A J, SEGALL-SHAPIRO T H, GLASSEY E, et al. Escherichia coli “Marionette” strains with 12 highly optimized small-molecule sensors[J]. Nature Chemical Biology, 2019, 15(2): 196-204.
[20]
WANG W Y, LI Y, WANG Y Q, et al. Bacteriophage T7 transcription system: an enabling tool in synthetic biology[J]. Biotechnology Advances, 2018, 36(8): 2129-2137.
Since its discovery in the 1970s, the T7 RNA polymerase (T7 RNAP) transcription system has been applied extensively as an effective tool in molecular biology because of its robust function in various hosts, including prokaryotic, eukaryotic and cell free systems. Recently, the T7 RNAP transcription system has emerged as a critical component for synthetic biology. The present paper summarizes the advances of the T7 RNAP transcription system in synthetic biology, including the recent progress of T7 RNAP structure and its cognate promoter and terminator and its application in cell free systems, logic gates and orthogonal genetic circuits.Copyright © 2018 Elsevier Inc. All rights reserved.
[21]
CHENG S-Y, LIN T H, CHEN P-T. Integration of multiple phage attachment sites system to create the chromosomal T7 system for protein production in Escherichia coli nissle 1917[J]. Journal of Agricultural and Food Chemistry, 2022, 70(33): 10239-10247.
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[25]
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There is a groundswell of interest in using genetically engineered sensor bacteria to study gut microbiota pathways, and diagnose or treat associated diseases. Here, we computationally identify the first biological thiosulfate sensor and an improved tetrathionate sensor, both two-component systems from marine Shewanella species, and validate them in laboratory Escherichia coli. Then, we port these sensors into a gut-adapted probiotic E. coli strain, and develop a method based upon oral gavage and flow cytometry of colon and fecal samples to demonstrate that colon inflammation (colitis) activates the thiosulfate sensor in mice harboring native gut microbiota. Our thiosulfate sensor may have applications in bacterial diagnostics or therapeutics. Finally, our approach can be replicated for a wide range of bacterial sensors and should thus enable a new class of minimally invasive studies of gut microbiota pathways.
[26]
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[27]
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We describe an isothermal, single-reaction method for assembling multiple overlapping DNA molecules by the concerted action of a 5' exonuclease, a DNA polymerase and a DNA ligase. First we recessed DNA fragments, yielding single-stranded DNA overhangs that specifically annealed, and then covalently joined them. This assembly method can be used to seamlessly construct synthetic and natural genes, genetic pathways and entire genomes, and could be a useful molecular engineering tool.
[28]
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Synthetic genetic sensors and circuits enable programmable control over the timing and conditions of gene expression. They are being increasingly incorporated into the control of complex, multigene pathways and cellular functions. Here, we propose a design strategy to genetically separate the sensing/circuitry functions from the pathway to be controlled. This separation is achieved by having the output of the circuit drive the expression of a polymerase, which then activates the pathway from polymerase-specific promoters. The sensors, circuits and polymerase are encoded together on a 'controller' plasmid. Variants of T7 RNA polymerase that reduce toxicity were constructed and used as scaffolds for the construction of four orthogonal polymerases identified via part mining that bind to unique promoter sequences. This set is highly orthogonal and induces cognate promoters by 8- to 75-fold more than off-target promoters. These orthogonal polymerases enable four independent channels linking the outputs of circuits to the control of different cellular functions. As a demonstration, we constructed a controller plasmid that integrates two inducible systems, implements an AND logic operation and toggles between metabolic pathways that change Escherichia coli green (deoxychromoviridans) and red (lycopene). The advantages of this organization are that (i) the regulation of the pathway can be changed simply by introducing a different controller plasmid, (ii) transcription is orthogonal to host machinery and (iii) the pathway genes are not transcribed in the absence of a controller and are thus more easily carried without invoking evolutionary pressure.
[30]
MA Y Y, ZHENG X R, LIN Y N, et al. Engineering an oleic acid-induced system for Halomonas, E. coli and Pseudomonas[J]. Metabolic Engineering, 2022, 72: 325-336. DOI:10.1016/j.ymben.2022.04.003
[31]
LIN T H, CHENG S-Y, LIN Y-F, et al. Development of the low-temperature inducible system for recombinant protein production in Escherichia coli nissle 1917[J]. Journal of Agricultural and Food Chemistry, 2024, 72(13): 7318-7325.
[32]
ABEDI M H, YAO M S, MITTELSTEIN D R, et al. Ultrasound-controllable engineered bacteria for cancer immunotherapy[J]. Nature Communications, 2022, 13: 1585. DOI:10.1038/s41467-022-29065-2.
Rapid advances in synthetic biology are driving the development of genetically engineered microbes as therapeutic agents for a multitude of human diseases, including cancer. The immunosuppressive microenvironment of solid tumors, in particular, creates a favorable niche for systemically administered bacteria to engraft and release therapeutic payloads. However, such payloads can be harmful if released outside the tumor in healthy tissues where the bacteria also engraft in smaller numbers. To address this limitation, we engineer therapeutic bacteria to be controlled by focused ultrasound, a form of energy that can be applied noninvasively to specific anatomical sites such as solid tumors. This control is provided by a temperature-actuated genetic state switch that produces lasting therapeutic output in response to briefly applied focused ultrasound hyperthermia. Using a combination of rational design and high-throughput screening we optimize the switching circuits of engineered cells and connect their activity to the release of immune checkpoint inhibitors. In a clinically relevant cancer model, ultrasound-activated therapeutic microbes successfully turn on in situ and induce a marked suppression of tumor growth. This technology provides a critical tool for the spatiotemporal targeting of potent bacterial therapeutics in a variety of biological and clinical scenarios.© 2022. The Author(s).
[33]
何燕斌, 齐亚坤, 黄霖霆, 等. 一种低温表达型T载体及其应用[J]. 生物工程学报, 2015, 31(12): 1773-1783.
HE Y B, QI Y K, HUANG L T, et al. An expression T-vector and its application at low temperatures[J]. Chinese Journal of Biotechnology, 2015, 31(12): 1773-1783.
[34]
WICHMANN J, BEHRENDT G, BOECKER S, et al. Characterizing and utilizing oxygen-dependent promoters for efficient dynamic metabolic engineering[J]. Metabolic Engineering, 2023, 77: 199-207. DOI:10.1016/j.ymben.2023.04.006.
Promoters adjust cellular gene expression in response to internal or external signals and are key elements for implementing dynamic metabolic engineering concepts in fermentation processes. One useful signal is the dissolved oxygen content of the culture medium, since production phases often proceed in anaerobic conditions. Although several oxygen-dependent promoters have been described, a comprehensive and comparative study is missing. The goal of this work is to systematically test and characterize 15 promoter candidates that have been previously reported to be induced upon oxygen depletion in Escherichia coli. For this purpose, we developed a microtiter plate-level screening using an algal oxygen-independent flavin-based fluorescent protein and additionally employed flow cytometry analysis for verification. Various expression levels and dynamic ranges could be observed, and six promoters (nar-strong, nar-medium, nar-weak, nirB-m, yfiD-m, and fnrF8) appear particularly suited for dynamic metabolic engineering applications. We demonstrate applicability of these candidates for dynamic induction of enforced ATP wasting, a metabolic engineering approach to increase productivity of microbial strains that requires a narrow level of ATPase expression for optimal function. The selected candidates exhibited sufficient tightness under aerobic conditions while, under complete anaerobiosis, driving expression of the cytosolic F-subunit of the ATPase from E. coli to levels that resulted in unprecedented specific glucose uptake rates. We finally utilized the nirB-m promoter to demonstrate the optimization of a two-stage lactate production process by dynamically enforcing ATP wasting, which is automatically turned on in the anaerobic (growth-arrested) production phase to boost the volumetric productivity. Our results are valuable for implementing metabolic control and bioprocess design concepts that use oxygen as signal for regulation and induction.Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.
[35]
ZHOU Y, KONG D Q, WANG X Y, et al. A small and highly sensitive red/far-red optogenetic switch for applications in mammals[J]. Nature Biotechnology, 2022, 40(2): 262-272.
[36]
WANG Z, DAI Y Q, AZI F, et al. Deciphering the crucial roles of the quorum-sensing transcription factor SdiA in NADPH metabolism and (S)-equol production in Escherichia coli nissle 1917[J]. Antioxidants, 2024, 13(3): 259.
[37]
VEISI K, FARAJNIA S, ZARGHAMI N, et al. Chaperone-assisted soluble expression of a humanized anti-EGFR ScFv antibody in E. coli[J]. Advanced Pharmaceutical Bulletin, 2015, 5(Suppl 1): 621-627.
[38]
YASAMUT U, THONGHEANG K, WEECHAN A, et al. Evaluating the ability of different chaperones in improving soluble expression of a triple-mutated human interferon gamma in Escherichia coli[J]. Journal of Bioscience and Bioengineering, 2024, 138(3): 232-238.
[39]
ROSANO G L, CECCARELLI E A. Recombinant protein expression in Escherichia coli: advances and challenges[J]. Frontiers in Microbiology, 2014, 5: 172. DOI:10.3389/fmicb.2014.00172
[40]
FATIMA K, NAQVI F, YOUNAS H. A review: molecular chaperone-mediated folding, unfolding and disaggregation of expressed recombinant proteins[J]. Cell Biochemistry and Biophysics, 2021, 79(2): 153-174.
The advancements in biotechnology over time have led to an increase in the demand of pure, soluble and functionally active proteins. Recombinant protein production has thus been employed to obtain high expression of purified proteins in bulk. E. coli is considered as the most desirable host for recombinant protein production due to its inexpensive and fast cultivation, simple nutritional requirements and known genetics. Despite all these benefits, recombinant protein production often comes with drawbacks, such as, the most common being the formation of inclusion bodies due to improper protein folding. Consequently, this can lead to the loss of the structure-function relationship of a protein. Apart from various strategies, one major strategy to resolve this issue is the use of molecular chaperones that act as folding modulators for proteins. Molecular chaperones assist newly synthesized, aggregated or misfolded proteins to fold into their native conformations. Chaperones have been widely used to improve the expression of various proteins which are otherwise difficult to produce in E. coli. Here, we discuss the structure, function, and role of major E. coli molecular chaperones in recombinant technology such as trigger factor, GroEL, DnaK and ClpB.
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