Part:BBa_K5377810

SAM self-induced production system

The BBa_K5377800& BBa_K5377830 are also our Best Composite Parts

pJ23106-SAM-I/IV variant riboswitch-lacI-pTrc-lacO-metK-metA-metB-metE-rrnB T1

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

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

  Illegal BamHI site found at 1397
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 4029
  Illegal AgeI site found at 1977
  Illegal AgeI site found at 4497
  Illegal AgeI site found at 6219
  
• 1000
  COMPATIBLE WITH RFC[1000]

Description

In order to self-induced production of S-Adenosyl methionine (SAM) without the need for human intervention, our team designed this part. This composite part integrates the SAM-I/IV variant riboswitch 'ON system' and a SAM synthesis operon. It is mainly used to detect subtle fluctuations in intracellular SAM concentrations and intelligently regulate the SAM synthesis pathways through SAM-I/IV variant riboswitch(Referred to as the 'SAM riboswitch' below. ). 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 SAM is low, the SAM 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 SAM.

Fig.1 The gene circuit of the SAM 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 a 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.

Congregibacter litoralis KT71 SAM-I/IV variant riboswitch

Introduction

The SAM-I/IV variant riboswitch in Congregibacter litoralis KT71 is a unique RNA regulatory element that controls genes involved in sulfur metabolism by binding S-adenosylmethionine (SAM), a vital metabolite in methylation and sulfur transfer reactions. Riboswitches are structured RNA molecules that can directly bind metabolites and modulate gene expression without the need for proteins.
The SAM-I/IV riboswitch is a hybrid of two distinct riboswitch classes: SAM-I and SAM-IV. SAM-I riboswitches, one of the most studied riboswitch classes, regulate genes responsible for SAM biosynthesis and sulfur metabolism in many bacteria. SAM-IV riboswitches are a more recently discovered class with some structural differences but similar function. ^[1] This variant combines characteristics from both classes, providing Congregibacter litoralis with flexible control over sulfur-related genes, ensuring efficient regulation in response to environmental changes.
Upon SAM binding, the SAM-I/IV riboswitch undergoes a conformational change, regulating transcription and inhibiting SAM biosynthesis when SAM levels are sufficient.

Function

In the absence of SAM, the riboswitch adopts a structure that allows the transcription of the SAM synthesis gene, promoting the production of SAM. However, when SAM binds to the riboswitch, it triggers a structural rearrangement that either terminates transcription prematurely depending on the organism and the regulatory mechanism.
By replacing the gene downstream of the riboswitch sequence with the lacI gene, the riboswitch directly regulates the expression of the repressor protein instead of controlling SAM biosynthesis.

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

Fig.3 Downregulation of sfgfp expression by the SAM riboswitch at different SAM 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.^[2]

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. ^[3]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.^[4]
We use it together with the trc promoter to co-regulate the expression of the downstream SAM synthesis operon, enabling SAM production to be controlled by the SAM riboswitch, thereby forming a complete riboswitch 'ON system'.

RBS&Operon

Introduction

We have designed an S-Adenosylmethionine (SAM) biosynthesis operon for expression in E.coli to achieve efficient production of SAM. The operon consists of four key genes: metK, metA, metB, and metE,along with the constitutive prokaryotic from Registry of Parts. Each gene encodes a specific enzyme that participates in the SAM biosynthetic pathway.
The SAM synthesis operon is constituted by four genes:
metK: This gene encodes S-adenosylmethionine synthetase, whitch catalyzes the formation of SAM from ATP and L-methionine.
metA: This gene encodes Homoserine O-succinyltransferase. The enzyme is involved in the first step of methionine biosynthesis. It converts L-homoserine into O-succinyl-L-homoserine, which is a precursor in the methionine biosynthesis pathway.
metB: This gene encodes Cystathionine gamma-synthase, which catalyzes the conversion of O-succinyl-homoserine into cystathionine, another intermediate in the methionine biosynthesis pathway.
metE: This gene encodes Cobalamin-independent methionine synthase that catalyzes the final step in the methionine biosynthesis pathway, converting homocysteine into methionine, which is then available for SAM synthesis.
(For more information on each individual RBS and gene of the SAM operon, see the following parts pages: BBa_B0035&BBa_K417000 for metK, BBa_B0034&BBa_K804004 for metA, BBa_B0029&BBa_K5377101 for metB,and BBa_B0032&BBa_K5377102 for metE.)
These enzymes' sequential actions form an effective metabolic pathway, enabling E.coli to synthesize more SAM.

Part Characterization

Functional Validation of Natural Riboswitch

You can find detailed information on the BBa_K5377100page.
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 SAM riboswitch, the fluorescence intensity decreased at SAM concentrations of 0.1 mM, 0.2 mM, 0.3 mM, and 0.4 mM, with a maximum reduction of 93% at 0.4 mM. (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 sfgfp 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 (0.3 mM SAM) 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 SAM riboswitch, the fermentation entered the stationary phase at 9 hours, and at this point, the fluorescence intensity was increased by 215% compared to the control. (The results are shown in Fig. 4.) This fully demonstrates that our approach is feasible!

Fig.4 metK SAM riboswitch ON-sfGFP up-regulated expression

L-SAM-ON: 0mM R-SAM-ON: 0.1mM

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

Riboswitch-Mediated Self-Induced SAM Synthesis

Introduction

To validate the feasibility of self-induced SAM production, we constructed a production strain containing this composite part sequence and used IPTG induced strain (with the same SAM 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 HPLC 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, Thermo Scientific™ UltiMate 3000 HPLC , 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 SAM titer was only 37.04 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 SAM, the fermentation titer reached 348.37 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 SAM production.

However, the efficiency of self-induced production still needs to be further improved. For example, in the case of SAM 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 SAM titers were 352.63, 386.43, 331.19 mg/L, respectively. The titer of the self-induced production strain was 348.37 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.