A Toolkit to Enable Hydrocarbon Conversion in Aqueous Environments

摘要

A sustainable auto regulating bacterial system for the remediation of oil pollutions was designed using standard interchangeable DNA parts (BioBricks). An engineered E. coli strain was used to degrade alkanes via β-oxidation in toxic aqueous environments. The respective enzymes from different species showed alkane degradation activity. Additionally, an increased tolerance to n-hexane was achieved by introducing genes from alkane-tolerant bacteria.

摘要

This work puts forward a toolkit that enables the conversion of alkanes by Escherichia coli and presents a proof of principle of its applicability. The toolkit consists of multiple standard interchangeable parts (BioBricks)⁹ addressing the conversion of alkanes, regulation of gene expression and survival in toxic hydrocarbon-rich environments.

A three-step pathway for alkane degradation was implemented in E. coli to enable the conversion of medium- and long-chain alkanes to their respective alkanols, alkanals and ultimately alkanoic-acids. The latter were metabolized via the native β-oxidation pathway. To facilitate the oxidation of medium-chain alkanes (C5-C13) and cycloalkanes (C5-C8), four genes (alkB2, rubA3, rubA4and rubB) of the alkane hydroxylase system from Gordonia sp. TF6^8,21 were transformed into E. coli. For the conversion of long-chain alkanes (C15-C36), theladA gene from Geobacillus thermodenitrificans was implemented. For the required further steps of the degradation process, ADH and ALDH (originating from G. thermodenitrificans) were introduced^10,11. The activity was measured by resting cell assays. For each oxidative step, enzyme activity was observed.

To optimize the process efficiency, the expression was only induced under low glucose conditions: a substrate-regulated promoter, pCaiF, was used. pCaiF is present in E. coli K12 and regulates the expression of the genes involved in the degradation of non-glucose carbon sources.

The last part of the toolkit - targeting survival - was implemented using solvent tolerance genes, PhPFDα and β, both from Pyrococcus horikoshii OT3. Organic solvents can induce cell stress and decreased survivability by negatively affecting protein folding. As chaperones, PhPFDα and β improve the protein folding process e.g. under the presence of alkanes. The expression of these genes led to an improved hydrocarbon tolerance shown by an increased growth rate (up to 50%) in the presences of 10% n-hexane in the culture medium were observed.

Summarizing, the results indicate that the toolkit enables E. coli to convert and tolerate hydrocarbons in aqueous environments. As such, it represents an initial step towards a sustainable solution for oil-remediation using a synthetic biology approach.

引言

石油污染是造成环境污染最严重的原因之一，对生态系统、企业和社区产生重大影响 ³.例如，需要解决方案来对抗加拿大阿尔伯塔省油砂尾矿水域产生的持续石油污染。在从油砂中提取石油的过程中，沥青（一种半固体氧化形式的石油）使用热回收技术去除，每桶油消耗约 3.1 桶水 ¹。受油污染的工艺用水主要来自当地河流，在沥青提取后储存在尾矿池中。需要更有效地回收工艺用水，以减少对淡水的吸收。为了促进沥青的开采并确保下游站点符合保护水生生态系统的水质准则，工艺用水处理正在迅速发展 ³。

为了处理有机化合物的污染，目前鼓励采用微生物的生物修复技术 ¹.烷烃是原油中最丰富的碳氢化合物家族，每个分子含有 5 至 40 个碳原子 ^{7， 21}。已知许多细菌通过末端甲基的顺序氧化降解不同长度的烷烃，首先形成醇，然后是醛，最后形成脂肪酸 ⁸。在这个 iGEM 项目中，表达和表征了来自不同生物体的几种酶，并通过 BioBrick 标准品和标准生物部分登记处提供。

经过充分研究的 Gordonia sp. TF6 烷烃羟化酶系统使用至少四种组分促进 C_{5-C 13} 烷烃和 C_{5-C 8} 环烷烃的初始氧化步骤：alkB2（烷烃 1-单加氧酶）、rubA3、rubA4（两种 rubredoxin 还原酶）和 RubB（rubredoxin 还原酶）^{8， 21}。据报道，长链烷烃（范围从 C₁₅ 到 C₃₆）的氧化由 ladA 进行，ladA 是一种来自热二硝酸杆菌 NG-80-2 ^{7、15、18、22} 的黄素蛋白烷烃单加氧酶。LadA 与黄素单核苷酸 （FMN） 形成催化复合物，利用原子氧进行氧化。这导致烷烃转化为相应的伯烷醇。醇类被醇和醛脱氢酶进一步氧化成脂肪酸，脂肪酸很容易进入β氧化途径 ^{7， 21}。来自嗜热细菌 Geobacillus thermoleovorans B23 的锌非依赖性醇脱氢酶使用 NAD⁺ 作为辅因子 ¹⁰ 将中链烷醇氧化成各自的烷醇。来自同一细菌的醛脱氢酶能够催化中链氧化中依赖于 NAD⁺ 的最后一步 ¹¹。

为了降低诱导成本并保持细菌系统的最佳增殖，对来自大肠杆菌的启动子 pCaiF 进行了表征。该启动子可以调节碳氢化合物降解途径组分的表达，并受 cAMP-Crp 水平的调节，而 cAMP-Crp 水平又取决于葡萄糖水平 ⁶。在环境中细胞外葡萄糖浓度高的情况下，细胞 cAMP （环腺苷单核苷酸磷酸盐） 水平通过抑制腺苷环化酶作为 PTS 介导的葡萄糖转运的副作用而较低。相反，在限制（低葡萄糖浓度）期间，cAMP 水平增加，Crp 与 cAMP 结合形成复合体 cAMP-Crp，该复合体结合 pCaiF 并激活下游组分的转录 ^{6， 14}。

野生型大肠杆菌只能耐受中等浓度的碳氢化合物。为了完成该工具包，必须解决对碳氢化合物的耐受性问题。已知几种耐有机溶剂的细菌可以在水溶剂两相系统中存活 ¹²。已知会增加耐受性的分子成分是促进蛋白质正确折叠的伴侣。来自堀越火球菌 OT3 的前折叠蛋白系统由蛋白质 phPFDα 和 phPFDβ 组成，被证明可以提高碳氢化合物耐受性 ¹⁷。

烷烃转化工具包是按照 BioBrick 原理构建的，该原理记录在标准生物第 ⁹ 部分注册表中。BioBricks 是含有特定功能插入片段的质粒，两侧有 4 个预定义的限制性位点。BioBrick 插件可以灵活扩展，从而构建具有新功能的生物系统。

方案

1. BioBrick Assembly

1. BioBricks from the Registry of Standard Biological parts are provided by iGEM headquarters. To construct a new BioBrick from existing BioBricks, digest the donor BioBrick (up to 1.0 μg) with the enzymes EcoRI and SpeI for positioning the donor part downstream of the acceptor part. Digest with XbaI and PstI for positioning the donor part upstream of the acceptor part. Add a third appropriate restriction enzyme that cuts in the backbone of the donor. Perform the digestions in a total volume of 20-25 ml with the appropriate buffer, according to the supplier (final concentration 1x). Use 5 units/μg DNA for the restriction enzymes.
2. Digest the acceptor BioBrick with either EcoRI and XbaI or SpeI and PstI.
3. Incubate the digestions for (at least) one hr at 37 °C. Inactivate the restriction endonucleases by heat, incubation at 80 °C for 10 min and centrifuge shortly.
4. Ligate the digested BioBrick parts (donor and acceptor) together. Since XbaI and SpeI generate compatible DNA ends, a mixed site is created that cannot be cut with any restriction enzyme resulting in a new 'combined' BioBrick that flanked by the 4 standard restriction sites. In the ligation mixture the final DNA concentration is preferably ~100 ng/μl. Perform the ligation reaction in a total volume of 10-15 ml with the T4 ligation buffer (final concentration 1x) and T4 ligase (1 unit/μg DNA).
5. Incubate the ligation mixture at 16 °C for at least 3 hr.
6. Perform transformation reaction with circa half of the ligation mix.
7. Confirm the BioBrick to be correct with sequencing.
8. To construct a new BioBrick from synthesized DNA, modify the genes for synthesis for optimal expression in E. coli by means of the JCat website tool (http://www.jcat.de/). Make further modifications in accordance with the requirements of the BioBrick standard. This standardization implies that every BioBrick is composed of a DNA sequence of interest preceded by a prefix and followed by a suffix. The prefix and suffix are sequences that contain predefined restriction sites, that are absent in the remaining plasmid sequence. These standard restriction sites make it possible to interchange and extend the BioBrick inserts flexibly ⁹. The prefix and suffix have the following sequences:

Name	Sequence	Comment
Prefix	5' GAATTCGCGGCCGCTTCTAG3'
	5' GAATTCGCGGCCGCTTCTAGAG 3'	If the following part is a coding sequence or any part that starts with "ATG"
Suffix	5' TACTAGTAGCGGCCGCTGCAG 3'

2. Alkane Conversion Resting Cell Assay, In Vivo

This assay was performed based on the method described by Fujii et al. (2004).

1. Culture E. coli cells expressing the Alkane Hydroxylase system (BBa_K398014) and cells carrying an empty vector (BBa_J13002) in 5 ml LB medium with appropriate antibiotics overnight.
2. Transfer 500 μl of the overnight culture into 50 ml of fresh LB (with antibiotic) and incubate until the cell turbidity reached an OD (optical density) of 0.3 at 600 nm.
3. Centrifuge for 10 min at 4,000 rpm and resuspend the pellet in 5 ml 0.1 M phosphate buffer (pH 7.4).
4. Centrifuge again for 10 min at 4,000 rpm and resuspend the pellet in 5 ml 0.1 M phosphate buffer now containing E2 salts and 0.66% v/v glycerol (nitrogen-deficient medium).
5. Measure the cell turbidity (OD₆₀₀).
6. Prepare cell-mixture aliquots of 6 ml in 25 ml closed-cap glass flasks and no-cell controls (E2 salts + 0.66% v/v glycerol).
7. Add 100 nmol of alkane to each flask.
8. Incubate the mixtures at 37 °C for 24 hr (shorten when higher rates are obtained).
9. Measure the OD₆₀₀ after incubation.
10. Extract the hydrocarbons in the culture media by ethyl acetate and determine hydrocarbon concentration in cell culture by gas chromatography (see protocol 3).
11. Calculate the degradation per unit of biomass by dividing the total amount of alkane converted by the total biomass present in each flask (convert OD₆₀₀ to dry weight). Dividing the result by the experimental duration yields the degradation activity per unit biomass per time unit. The average is taken from three individual runs.

3. Alkane Conversion Enzyme Assay, In Vitro

This assay was performed essentially according to the method described by Li et al. (2008).

1. Culture E. coli cells expressing the ladA gene (BBa_K398017) and cells carrying an empty vector (BBa_J13002) in 50 ml LB medium with appropriate antibiotics overnight.
2. Transfer 500 μl of the overnight culture into 50 ml of fresh LB (with antibiotic) and incubate until the cell turbidity reached an optical density of 0.6 at 600 nm.
3. Centrifuge 10 min at 4,000 rpm (4 °C) and resuspend the pellet in 5 ml of 50 mM Tris buffer.
4. Sonicate (Cell disrupter, LA Biosystems) the cells at 40% duty cycle with an output control of 4, keep the solution on ice for the entire duration.
5. Centrifuge the resulting mixture for 5 min at 4,000 rpm at 4 °C to remove the cell debris. Transfer the supernatant to a fresh vial.
6. Determine the total protein concentration of the cell extracts by Bradford assay. Note: Use glass vials to prevent the increase of background and/or loss of protein.
7. Prepare a 100 ml mixture containing 0.1% v/v alkane and 50 mM Tris-HCl buffer.
8. Heat the mixture at 100 °C for 5 min. Note: Perform this step only for medium-long chain alkanes with high boiling point. (e.g. C₁₆: 287 °C).
9. To achieve an optimal solubility of the alkane, sonicate for 1 min while still warm until a homogenous, viscous mixture is obtained.
10. Add 1mM of NADH, 1 mM FMN, 1 mM MgSO₄ and 0.01 v/v Triton X-100.
11. Prepare 6 ml aliquots in 25 ml closed-cap flasks.
12. Add adequate amounts of cell extract (depends on Bradford assays, final concentration of 5 mg protein/l). Prepare a no-protein control.
13. Incubate at 60 °C (for optimal enzymatic activity) for 24 hr.
14. Extract the hydrocarbons in the culture media by ethyl acetate and determine hydrocarbon concentration in cell culture by gas chromatography (see protocol 3).
15. Calculate the degradation per unit of biomass by dividing the total amount of alkane converted by total protein added to each flask (by Bradford calibration curve). Further dividing by experiment duration yields the degradation activity per unit of cellular protein per time unit. The average is taken from three individual runs.

4. Ethyl Acetate Hydrocarbon Extraction and Concentration Measurements

1. Alkanes are extracted from the aqueous solution by adding 2.5 ml ethyl acetate (apolar solvant) to 6 ml experimental solution. An internal standard is added to the solvent at a concentration of 0.1 % (v/v). The standard (e.g. cyclo-decan) varied depending on the expected range of the peaks of interest.
2. Optional: Add Triton X-100 to the aqueous mixture and centrifuge the samples for 10 min at 4,000 rpm in order to get a proper bi-phasic system.
3. Vortex the mixture for 5 sec (1,500 rpm) and incubate at room temperature until the two phases separate.
4. Remove a maximal amount of the organic layer (top) and dry the solvent using anhydrous magnesium sulphate.
5. Remove MgSO₄ by filtration (0.2 μm) and transfer the filtrate into gas chromatograph vials for measurements, or store at -20 °C.
6. Determine the concentration by gas chromatography using a CP-SIL 5CB column (length = 5 m). Inject 10 μl of sample in split mode (1:10, 230 °C). Set the column gas flow to 1 ml/min (Helium). The following oven temperature program is used:

   Rate	Temperature [°C]	Time [min]
   0	50	7.5
   50	90	1.0
   50	110	2.0
   50	130	2.0
   50	145	2.0
   50	160	2.0
   50	170	2.0
   50	185	2.0
   50	210	2.0
   50	250	2.0
   50	320	2.0

7. Integrate peaks and correct the concentrations with respect to the internal standard.

5. Alcohol/aldehyde Dehydrogenase Activity Assay

This assay was performed essentially according to the method described by Kato et al. (2010).

1. Culture E. coli cells expressing the ADH (BBa_K398018) or ALDH (BBa_K398030) gene and cells carrying an empty vector (BBa_J13002) in 50 ml LB medium with appropriate antibiotics overnight.
2. Transfer 500 μl of the overnight culture into 50 ml of fresh LB (with antibiotic) and incubate until the cell turbidity reached an optical density of 0.6 at 600 nm.
3. Centrifuge 10 min at 4,000 rpm at 4 °C and resuspend the pellet in 5 ml of 50 mM Tris buffer.
4. Sonicate the cell solution at 40% duty cycle with an output control of 4 (keep the solution on ice during the sonication).
5. Centrifuge the resulting mixture for 5 min at 4,000 rpm at 4 °C to remove cell debris.
6. Determine the total protein concentration of the cell extracts by Bradford assay (Note: use a glass vial to prevent protein binding).
7. Load a 96 well plate with 180 μl 57 mM glycine buffer containing NAD (final concentration 1 mM, pH 9.5) in each well.
8. Add 5 μl of the alcohol (aldehyde) to be tested to the wells. Note: Heat long chain alcohols before the start of the assay to have a liquid. For each alcohol (aldehyde) a control without substrate should be added (negative control). In addition, prepare a blank containing a mixture of buffer and the substrate without cell extract.
9. Preheat the plate of the plate reader (Tecan Magellan v7.0) for 15 min at 37 °C to allow equilibration of the system.
10. Add adequate amounts of cell extract (depends on Bradford assays, final concentration of 5 mg protein/l). Prepare a no-protein control.
11. Measure the NADH production using a spectrophotometer at a wavelength of 340 nm every 2-3 min for 1 hr at 37 °C.
12. Calculate the NADH production rate from the slope of the OD (340 nm). Take into account the light path length and the extinction coefficient of NADH of 6220 M^-1cm^-1. Divide the NADH production rate by the total amount of protein added to express the activity of the dehydrogenase reaction in the cell extract (U/mg whole cell protein). Calculate the mean and the standard deviation from three independent runs.

6. pCaiF Characterization

1. Culture E. coli cells expressing the pCaiF-GFP construct (BBa_K398331) and cells carrying the promoter alone (BBa_K398326) overnight in 5 ml LB medium the appropriate antibiotics overnight.
2. Inoculate 5 ml M9 containing 10 g/l glucose and antibiotics with 50 μl of the overnight culture and grow overnight.
3. Subculture 50 μl of the overnight outgrowth into 5 ml of fresh M9 with10 g/l and incubate until the cell turbidity reached an optical density of 0.2 at 600 nm.
4. Load a 96 well plate with 100 μl of fresh M9 medium containing the desired amount of carbon source for testing in each well. Perform triplicate experiments for statistical evaluation and add the respective negative controls (wild-type E. coli K12).
5. Add 5 μl of the overnight culture to the medium containing wells.
6. Measure the growth curve (OD₆₀₀) and GFP fluorescence (485 nm excitation and 520 emission) using a plate reader every 10 min for 18 hr at 37 °C with constant shaking.
7. Calculate the growth rate and the specific GFP content from the respective measurements and compare to the control. Calculate the mean and the standard deviation of at least three independent experiments.

7. Tolerance Assay

1. Culture E. coli expressing PhPFDα and β gene (BBa_K398406) overnight in 5 ml LB medium and the appropriate antibiotics. E. coli expressing ladA gene (BBa_K398017) is used as negative control.
2. Subculture 10 μl of the overnight outgrowth into fresh 5 ml of LB (with antibiotic) and incubate until the cell turbidity reached an optical density of 0.3 to 0.4 at 600 nm.
3. Dilute the cultures with fresh medium until an OD₆₀₀ of 0.1 is reached.
4. Load a 96 well plate with 180 μl M9 medium containing the appropriate antibiotics and the proper final concentration of the toxic compound (e.g. 0, 4, 8, 10% of n-hexane) in triplicate. Because alkane-water mixtures could lead to two-phase systems it is essential to have appropriate controls on the plate (e.g. different strains and the respective blank experiments).
5. Add 20 μl of the culture (control) into the wells.
6. Measure the biomass concentration (OD₆₀₀) using a plate reader every 10 min for 24 hr at 37 °C with constant shaking.
7. Calculate the growth rates from the respective measurements for the different agent concentrations and compare to the negative control. Calculate the mean and the standard deviation of at least three independent runs.

8. Homolog Interaction Mapping

1. The application HIM was developed to perform protein queries on a PostgreSQL server running the STRING database (free for academic use) ²⁰. The homologue interaction mapping application identifies interacting proteins in the original host organism using the STRING database. The sequences of the respective interacting genes are used in a BLAST search to find homologous genes in the target organism. Cytoscape ⁴ is used to visualize the result of the mapping. The HIM software tool can be downloaded at: https://github.com/jcnossen/InteractionHomologMapping.
2. To perform a mapping, (1) enter the BioBrick ID and the application will automatically download the part sequence data from the Registry of Standard Biological Parts ⁹, or (2) enter (paste) the sequence data in the application.
3. Use the STRING Database website to find the STRING protein ID for the entered amino acid sequence.
4. A protein with high homology is determined using BLAST. Subsequently the application lists each known interacting protein in the source organism and searches for homologs in the host organism (e.g. E. coli).
5. Export resulting putative interaction list to text or Cytoscape.

结果

烷烃转化率

使用静息细胞测定和酶活性测量评估从烷烃到相应脂肪酸的三个氧化步骤的活性。结果在途径反应 （1） 烷烃羟化酶、（2） 醇脱氢酶和 （3） 醛脱氢酶之后呈现><。 第一步，为中链和长链烷烃构建不同的质粒。质粒 BBa_K398014 包含用于中链烷烃氧化的烷烃羟化酶 （AH） 系统的四个基因：alkB2、rubA3、rubA4 和 rubB，受单个组成型启动子 （BBa_J23100） 的控制。作为阴性对照，使用带有质粒 BBa_J13002 的大肠杆菌 K12。使用 n...

讨论

The BioBrick principle is used to construct a chassis for the degradation of alkanes and a proof of principle for the single components of the toolkit was obtained. Several assays are proposed to measure the in vivo and in vitro activity of alkane degrading pathway enzymes. The presented work successfully demonstrates a number of methods that can be used to determine enzyme activities and expression in the host organism E. coli after implementation of suitable BioBricks. Furthermore, it is show...

材料

本文使用的材料清单

姓名	公司	目录编号	评论
大肠杆菌 K12	New England Biolabs	C2523H
辛烷	Fluka	74822
十六烷	Fluka	52209
辛醇-1	Fluka	95446
十二烷醇-1	Sigma-Aldrich	126799
己烷	Sigma-Aldrich	296090
NADH	Sigma-Aldrich	N4505
FMN	Sigma-Aldrich	F2253
MgSO4	J.T. Baker Casno	7487 889
Triton X-100	Sigma-Aldrich	T8787
T4 连接酶	New England Biolabs	M0202L
气相色谱仪
细胞破碎机	LABiosystems	CD-019
分光光度计	Amersham pharmacia	spec 2000
读板机	Tecan Group Ltd.	Magellan v7.0
培养箱	Innova，44
BioBrickTM K398014：BBa_J23100-BBa_J61100-alkB2-BBa_J61100-rubA3-BBa_J61100-rubA4- BBa_J61100-rubB	代尔夫特理工大学生物技术系或标准生物部件	注册处BBa_K398014	烷烃羟化酶系统 抗性：氯霉素
BioBrickTM K398027：BBa_R0040-BBa_B0034-ladA	代尔夫特理工大学生物技术系或标准生物部分	注册处BBa_K398027	ladA 蛋白质生成器 抗性：氯霉素
BioBrickTM K398018：BBa_J23100-BBa_J61101-ADH	代尔夫特理工大学生物技术系或标准生物部分	注册处BBa_K398018	ADH 生成器 抗性：氯霉素
BioBrickTM K398030：BBa_R0040-BBa_B0034-ALDH	代尔夫特理工大学生物技术系或标准生物部件	登记处BBa_K398030	ALDH 生成器 抗性：氯霉素
BioBrickTM K398326： pCaiF	代尔夫特理工大学生物技术系或标准生物部分登记	BBa_K398326	pCaiF 启动子 抗性：氯霉素
BioBrickTM K398331： pCaiF-BBa_B0032-BBa_I13401	代尔夫特理工大学生物技术系或标准生物部分登记	BBa_K398331	pCaiF 测量设备 抗性：氯霉素
BioBrickTM K398406：BBa_J23002-BBa_J61107-phPFDα-BBa_J61107-	代尔夫特理工大学生物技术系或标准生物部分登记	BBa_K398406	溶剂耐受性集群 耐性：氯霉素