Part:BBa_K5377830

TPP self-induced production system Ⅰ

This is one of ZJUT-China 2024's Best Composite Parts

pJ23106-thiM TPP riboswitch-lacI-pTrc-lacO-thiL-thiC-thiM-rrnB T1

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 2829
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 2829
  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 2829
  Illegal BglII site found at 3266
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 2829
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 2829
  Illegal AgeI site found at 1824
  Illegal AgeI site found at 3156
  Illegal AgeI site found at 3387
  
• 1000
  COMPATIBLE WITH RFC[1000]

Description

In order to self-induced production of TPP(Thiamine Pyrophosphate) without the need for human intervention, our team designed this part. This composite part integrates the thiM TPP riboswitch 'ON system' and a TPP synthesis operon. It is mainly used to detect subtle fluctuations in intracellular TPP concentrations and intelligently regulate the TPP synthesis pathways through thiM TPP riboswitch. By adding the lacI gene after the riboswitch sequence and inserting the lacO gene before the operon, we transformed the riboswitch from an 'OFF system' that is unfavorable for production into the 'ON system' that we need.
When the concentration of the TPP is low, the thiM TPP riboswitch remains in its ON state, allowing the downstream lacI gene to produce a repressor protein that binds to the lacO operator, inhibiting the expression of the small molecule synthesis operon. As the ligand concentration rises and reaches a specific threshold, the riboswitch shifts to the OFF state, stopping the production of the repressor protein. This enables normal expression of the operon, leading to significant synthesis of the small molecule TPP.

Fig.1 The gene circuit of the TPP self-inducing production system.

Usage and Biology

pJ23106

Introduction

The J23106 promoter is a moderate-strength constitutive promoter from the BBa_J23100 series, commonly used in synthetic biology for continuous gene expression without the need for external inducers. It is widely applied in bacterial systems, particularly in E. coli (Escherichia coli), to regulate the expression of reporter genes or enzymes in metabolic pathways. As part of a family of promoters with varying strengths, J23106 provides a flexible option for researchers to fine-tune the level of gene expression in their experiments.

thiM TPP riboswitch

Introduction

The thiM TPP riboswitch is a regulatory RNA element that specifically binds TPP (thiamine pyrophosphate) and controls the expression of genes involved in thiamine (vitamin B1) biosynthesis and transport, such as the thiM gene. This riboswitch is present in E. coli, functioning at either the transcriptional or translational level.^[2] Upon binding to TPP, the riboswitch undergoes a conformational change that represses gene expression by either terminating transcription or blocking translation initiation, depending on the organism and regulatory context.

Function

In the absence of TPP, the riboswitch adopts a structure that allows the transcription or translation of the thiM gene, promoting thiamine biosynthesis. When TPP is abundant, it binds to the riboswitch, triggering a structural rearrangement that halts the production of enzymes required for thiamine biosynthesis.^[3] This regulatory mechanism is highly conserved and plays a crucial role in maintaining proper cellular thiamine levels.
By replacing the thiM gene downstream of the riboswitch sequence with the lacI gene, the riboswitch directly regulates the expression of the repressor protein instead of controlling TPP biosynthesis.

Fig.2 The secondary structure of the TPP riboswitch. (From Wikipedia)

Fig.3 Downregulation of sfgfp expression by the thiM TPP riboswitch at different TPP concentrations.

lacI

Introduction

LacI, coding for lac repressor, is an usual basic part regularly used for repressing transcription. The lac repressor is a DNA-binding protein that inhibits the expression of genes coding for proteins involved in the metabolism of lactose in bacteria. These genes are repressed when lactose is not available to the cell, ensuring that the bacterium only invests energy in the production of machinery necessary for uptake and utilization of lactose when lactose is present. When lactose becomes available, it is converted into allolactose, which inhibits the lac repressor's DNA binding ability, thereby increasing gene expression. [BBa_K2963005]
The lac repressor could bind with the downstream lacO gene. In our project, we use it to transform the riboswitch from an 'OFF system' into 'ON system'.

pTrc

Introduction

The trc promoter is a strong hybrid promoter used extensively for high-level gene expression in E. coli. It combines elements of the trp (tryptophan) promoter and the lac (lactose) promoter to create a versatile system for regulated gene expression.^[3]

lacO

Introduction

LacO (the lac operator) is a DNA sequence within the lac operon of E. coli that plays a crucial role in the regulation of gene expression. It serves as the binding site for the lac repressor protein, encoded by the lacI gene. When the lac repressor is bound to the lacO operator, it prevents RNA polymerase from transcribing the downstream genes.^[4]In the presence of an inducer such as allolactose or IPTG, the lac repressor undergoes a conformational change, releasing its grip on the operator, allowing transcription to proceed.^[5]
We use it together with the trc promoter to co-regulate the expression of the downstream TPP synthesis operon, enabling TPP production to be controlled by the thiM TPP riboswitch, thereby forming a complete riboswitch 'ON system'.

RBS&Operon

Introduction

We have designed a TPP (Thiamine Pyrophosphate) synthesis operon for expression in E. coli to achieve efficient production of TPP. The operon consists of three key genes: thiL, thiC and thiM, along with the constitutive prokaryotic RBS from Registry of Parts. Each gene encodes a specific enzyme that participates in the TPP biosynthetic pathway.
The TPP synthesis operon is constituted by three genes:
thiL: The thiL gene encodes thiamine-monophosphate kinase, an enzyme responsible for phosphorylating thiamine monophosphate (TMP) into thiamine pyrophosphate (TPP), the active coenzyme form of vitamin B1. ThiL is essential in the final step of the TPP biosynthesis pathway, converting TMP into the biologically active TPP.
thiC: The thiC gene encodes phosphomethylpyrimidine synthase, an enzyme that catalyzes a crucial step in the biosynthesis of the pyrimidine moiety of thiamine. It converts 5-aminoimidazole ribotide (AIR) into 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate, a key intermediate in thiamine production.
thiM: The thiM gene encodes hydroxyethylthiazole kinase, an enzyme that catalyzes the phosphorylation of 4-methyl-5-(2-hydroxyethyl)thiazole, a precursor in the thiazole biosynthetic pathway. This step is important for the production of the thiazole moiety of thiamine, contributing to the overall biosynthesis of TPP.
(For more information on each individual RBS and gene of the TPP operon, see the following parts pages: B0035&K5377301 for thiL, B0034&K5377302 for thiC and B0029&K5377303 for thiM.)
These enzymes' sequential actions form an effective metabolic pathway, enabling E. coli to synthesize more riboflavin.

Part Characterization

Functional Validation of Natural Riboswitch

You can find detailed information on the BBa_K5377000page.
We successfully validated the OFF function of the natural riboswitches. Our team constructed the riboswitch 'OFF system' plasmid by using Gibson Assembly to insert the riboswitch fragment and sfgfp fragment into the pHG101 plasmid. This was then transformed into the E.coli MG1655 strain for fermentation.
For the ribB FMN riboswitch, the fluorescence intensity decreased by 72% at FMN concentrations of 0.2 mM, 0.4 mM, 0.6 mM, and 0.8 mM, respectively. (The results are shown in Fig. 3.)

Functional Reversal of Natural Riboswitch

Based on the 'OFF system' plasmid, we added the lacI and lacO components along with the Trc promoter to preliminarily construct the riboswitch 'ON system'.
Also, we successfully achieved the functional reversal of natural riboswitches, thereby realizing the ON function regulated by the riboswitches. In our previous experiments, we had already validated the OFF function of the natural riboswitches and observed a significant downregulation in GFP expression (i.e., fluorescence intensity) when appropriate concentration of small molecule ligands was added. Based on the results from the OFF function validation, we selected an appropriate concentration of the ligand (0.6mM FMN) to add.
Our team obtained encouraging fermentation results: The fluorescence intensity of the culture broth was significantly upregulated after the addition of the small molecule ligand compared to the control without the ligand. For the ribB FMN riboswitch, the fermentation entered the stationary phase at 9 hours, and at this point, the fluorescence intensity was increased by 325% compared to the control. (The results are shown in Fig. 4.) This fully demonstrates that our approach is feasible!

Fig.4 rib FMN riboswitch ON-sfGFP up-regulated expression

Next, introduce the RF synthesis operon sequence to the plasmid.

Riboswitch-Mediated Self-Induced Riboflavin Synthesis

Introduction

To further validate the feasibility of self-induced riboflavin production, we constructed a production strain containing this composite part sequence and used IPTG induced strain (with the same RF synthesis operon) as a control.

Methods

Add 200 μL of activated bacterial culture in flasks which contain 100 ml of M9 medium. Four control groups were set up: one group without IPTG induction and three groups induced with 0.5 mM IPTG at OD₆₀₀ values of 0.05, 0.5, and 1, respectively. After the fermentation started, samples were taken every 3 hours. The cells were sonicated, centrifuged, and the supernatant was collected for OD₄₄₄ absorbance detection. The concentration of the samples was calculated based on a standard curve plotted from the reference samples.

Material and Device

M9 medium, Thermo Scientific™ IPTG, S23A Visible Spectrophotometer from Shanghai Lengguang Technology Co., Ltd. , etc.

Results

We successfully achieved self-induced production of the product. For non-self-induced production strains, in the absence of an inducer, the target operon exhibited only minimal leaky expression, resulting in very low levels of fermentation product titer. For example, the riboflavin titer was only 19.86 mg/L. In contrast, the self-induced expression system, without any manual intervention, achieved self-induced synthesis of the product, with a significant increase in product concentration compared to the control. For riboflavin, the fermentation titer reached 414.31 mg/L. (The results are shown in Fig. 5.) This demonstrates that it is entirely feasible to use changes in product concentration during fermentation for self-induced production!

Fig.5 Time profiles of riboflavin production.

However, the efficiency of self-induced production still needs to be further improved. For example, in the case of riboflavin production, the titer of the manually induced system is significantly affected by the timing of inducer addition. When IPTG was added at OD₆₀₀ values of 0.05, 0.5, and 1, the riboflavin titers were 413.20, 484.30, 391.28 mg/L, respectively. The titer of the self-induced production strain was 414.31 mg/L, comparable to the titer obtained when the inducer was added at OD₆₀₀ = 0.05. This also suggests that there is room for optimizing the threshold and sensitivity of the riboswitch, which may be a direction to pursue in the future.