Our project is trying to offer an environmentally friendly biofertilizer via synthetic biology to solve the safety issues arising by the widespread use of herbicide. An engineered Escherichia coli were constructed to produce a novel herbicide, aspartic acid and extracellular polysaccharide (EPS) under blue light, which would be controllably released into the soil at a high temperature of 42°C, avoiding overuse of herbicides and possible residue, while promoting water retention and sand fixation by EPS.
Our engineered strain harbors a precursor plasmid that converts the glucose to the key precursor GPP, and a multiple functional plasmid to synthetize herbicide and EPS under the control of blue-light which is commonly used in the greenhouse. The controllable synthesis and release of herbicide and EPS of our system will maximize their effects and contribute to the environment and society. The multiple functions of our system have been validated individually and step by step in our lab work.
FIG 1. Scheme of our project
The precursor synthesis circuit is responsible for the GPP synthesis from glucose via MVA pathway. As multiple genes are expressed, the metabolic stress should be one of the considerations. Thus based on the previous work by 2019-XJTU-China (BBa_K3052010), we replaced the replicon P15Aori (5-10 copies) with the lower copy one oriV+trfA (1-3 copies). RT-qPCR showed that the transcription level of key enzyme GPPS was significantly increased (almost 3 fold) after the replacement of lower copy number replicons. The reduced metabolic pressure will lead to the improved cell growth and metabolism, also benefiting the protein expression.
FIG 2 The precursor synthesis circuit and replicon optimization strategy
FIG 3 RT-qPCR of GPPS in strains harboring plasmid with different ori
Compared to chemical induction, the light induction is more fine-tuned and spatial controllable. In order to achieve blue-light induction, we replace the arabinose binding and dimerization domain with blue-light responsive VVD domain, generating VVD-AraC fusion protein, which will dimerization under light and promote the downstream PBAD promoter. We tested three promoters for the expression of VVD-AraC fusion protein, native Pc, J23101 and porin promoter was selected, and porin promoter was demonstrated to be the most efficient one for sfGFP expression.
Meanwhile, a weak blue light induction system was developed in our project to test the efficiency of our system with a light intensity of 5W/m2. Finally, the engineered strain harboring pVVDH-porin showed constant increase of sfGFP intensity under blue light, demonstrating its good sensitivity, and dynamic response. The induction effect of blue-light was also confirmed by confocal, and numerous cells with green fluorescence were observed in the microscopy.
FIG 4 The blue light-inducible circuit with responsive VVD-AarC fusion protein
FIG 5 sfGFP detection of three strains with different promoters by self-made weak light inducing system
FIG 6 Change of sfGFP with time of strain with PVVDH-porin plasmid
FIG 7 Engineered cells was observed to show green fluorescence after blue-light induction
The AA sythesis circuit mainly contains astABC gene cluster for the synthesis of aspartic acid, under IPTG-inducible PlacUV5 promoter. It will convert the FPP generated by the precursor plasmid to aspartic acid. The broad-host-vector pBBRMCS1 is used as the backbone.
FIG 8 AA synthesis pathway from GPP and AA synthesis circuit
The astABC gene cluster was synthetize and amplified, and ligated to pMCS1 vector by Golden gate, and the plasmid containing AA gene cluster was constructed as demonstrating by colony PCR (FIG 9).
FIG 9 AA amplification and colony PCR verification
Due to the limitation of time and plant experiment, we are able to test the AA yield as well as its herbicide activity. But according to the literature, 100uM of AA is able to kill plant, exhibiting a higher activity than the commercial available herbicide glyphosate. Our primary work on the novel herbicide aspartic acid will benefit its wide applications in the future.
FIG 10 Activity test of AA conducted by Yan et al
The synthesis circuit for exopolysaccharides (EPS) was successfully constructed, including the EPS synthesis gene pmgA and galU under the control of Ptrc promoter (FIG 11). Previously EPS synthesis was also reported by team XJTU-iGEM in 2020, yet the yield was not satisfying. Our improvement on this issue included:
a) change the low efficient P43 promoter to strong inducible PlacUV5 promoter, to make the product synthesis more controllable;
b) replicon optimization to a high-copy number one pRO1600 ori.
Our modified plasmid was successfully constructed as verified by colony PCR and sequencing (FIG 11). The expression of EPS synthetic genes galU and pgmA was detected by RT-qPCR, which revealed increased transcriptional levels for both galU and pgmA gene. After IPTG induction, the expression levels of galU and pgmA gene were 3.4 and 2.8 fold compared to that of EE plasmid constructed by team 2020-XJTU-China.
As a result, the EPS yield of modified plasmid was also increased by almost 3 fold when tested by anthrone sulfuric acid method. Collectively, compared to the EE plasmid, our EPS synthesis plasmid showed higher protein expression and EPS yield, demonstrating the improvement after promoter and replicon optimization.
FIG 11 Optimization strategies and colony PCR verification
FIG 12 RT-qPCR result of EPS synthetic genes
FIG 13 EPS yield of three strains harboring EE plasmid and our modified plasmid 4
We designed the temperature-controlled suicide circuit for the biosafety and project feasibility. A novel lytic gene was found and constructed under the temperature sensitive Pλ promoter, to achieve the temperature control of bacterial lysis and product release when the temperature is 42°C or higher. Our plasmid 5 was successfully constructed as verified by colony PCR and sequencing.
The suicide verification clearly demonstrated the cell growth was significantly inhibited after heat at 42°C compared to the strain without lysis gene. And about 9% of whole cell population was retained after heat. Our project provides an alternative of lysis protein for MazF (BBa_K302033).
FIG 14 The temperature-controlled suicide circuit and colony PCR verification
FIG 15 Heat-triggered suicide test
Collectively, the multiple functions of our engineered bacteria have been validated individually by different gene circuits, which confirmed the functionality of blue-light induction system and temperature-controlled suicide system, as well as the synthesis of AA precursor pGPP and EPS. With the combination of these functions, our engineered strain can be constructed and improved for the implementation in the real greenhouse in the future. (See implementation part)
Although cell lysis reduces the population temporarily, about 10% of the engineered bacteria will escape the lysis process and recover, which will facilitate a new round of controlled production and release of herbicides and EPS.