Overview

The leguminous-nodule symbiosis system possesses a natural microaerobic environment. At the same time, rhizobia can respond sensitively to changes in environmental conditions, making it the top choice for an efficient biological microanaerobic synthesis platform.

We chose Sinorhizobium fredii CCBAU45436 as chassis, building up a set of regulation module in response to the changes of nitrogen and oxygen. Meanwhile, we selected genes encoding PUFA synthase and dipeptidyl aldehyde to preliminarily demonstrate the feasibility of the “microaerobic factory". In order to improve its efficiency, we reprogrammed the metabolism by adjusting the expression of relevant genes and designed the suicide circuit to avoid chassis leakage.

Here are three main parts of our design:

• PHB deletion

In order to increase the production of LC-PUFA, we deleted phaC2 gene in the genome of Sinorhizobium fredii CCBAU45436 by homologous recombinant double exchange. The lack of phaC2 blocks the synthesis of PHB and accumulates its substrate acetyl-coenzyme A, which is also the substrate of synthesizing LC-PUFA.


Figure1 The model of PHB deletion
• Regulation and synthesis module

The regulation module plays the central role in the whole synthesis circuit, which enables the downstream synthesis module to respond to nitrogen and oxygen. We selected dipeptide aldehyde synthesis genes and pfa biosynthetic gene clusters to demonstrate the ability of synthesizing complex secondary metabolites, preliminarily validating the feasibility of the regulation module.


Figure2 The model of synthesis module
• Suicide circuit

To prevent chassis escape and gene leakage, we designed a biosafety protection mechanism based on the CRISPRi system and the TA (toxin-antitoxin) system, which also responds to different nitrogen and oxygen conditions.

Module 1: PHB deletion

PHB (Poly-β-hydroxybutyrate) is an endogenous metabolite of Sinorhizobium fredii CCBAU45436 with high expression. phaC2 encodes the key enzyme in the synthesis of PHB, PHB synthase (PHB-synthase). phaC2 deletion causes decline of PHB synthesis in the chassis, so that the content of acetyl-CoA can relatively increase, providing sufficient substrate for the synthesis of PUFA.

To delete phaC2 gene, we select 500 bp upstream and downstream of phaC2 gene as homologous sequences and constructed the plasmid. We transferred it into chassis Sinorhizobium fredii CCBAU45436 by tri-parental mating, realizing the deletion of phaC2 gene by homologous recombination with the corresponding sequences.

Module 2: Regulation and Synthesis Module

Given the microaerobic environment of the symbiotic system between rhizobia and roots, we design a regulation module that responds to changes of nitrogen and oxygen concentration in the environment.

glnK promoter and nifH promoter are endogenous promoters of the chassis that respond to the changes of nitrogen and oxygen content. glnK promoter, an inducible promoter, will be activated under low nitrogen conditions and then initiate the expression of downstream genes; nifH promoter, an inducible promoter, will be activated under low oxygen conditions and then initiate the expression of downstream genes. Moreover, we use a non-cleavage-active Cas12k protein in the CRISPRi system, which reversibly binds to target sequences under the guidance of sgRNA and does not cleave them. Signaling is used to achieve binding and dissociation of Cas12k from downstream genes, leading to gene expression and repression. This module introduces the CRISPRi system to form the "AND gate" regulation mode, which realizes more accurate regulation of downstream genes.

In the following two states: in high oxygen and low nitrogen which indicate rhizobia are free, and in low oxygen and low nitrogen which indicate rhizobia perform nitrogen-fixing function in nodule, synthesis module is inhibited. The glnK promoter is activated by low nitrogen and initiates the expression of sgRNA-GFP, which guides the constitutively expressed Cas12k to bind to the gfp gene sequences and blocking the transcription of synthetic gene.

And in the later stage of plant growth, supplemental application of nitrogen fertilizer elevates the nitrogen concentration in the soil, which decreased the activity of glnK promoter. The expression of sgRNA-GFP decreases as a result. And Cas12k is unable to effectively block downstream gene transcription, thus realizing the expression of downstream synthetic gene initiated by the nifH promoter under hypoxic conditions. If the rhizobia escape to the outside of the nodule at this time, which exposed to high nitrogen and high oxygen environment, the activity of nifH promoter will be weakened, and it could not effectively initiate the downstream synthetic gene expression.

We first selected the dipeptide aldehyde synthesis gene bgc33-sfp to initially verify the validity of the regulation module. Meanwhile, bgc33-sfp encodes a nonribosomal peptide synthetase (NRPS) that catalyzes the synthesis of dipeptide aldehydes, a type of small molecule compounds with high potential for use in medicine but extremely susceptible to oxidation. Subsequently, we selected a larger pfa biosynthetic gene cluster from Aetherobacter fasciculatus SBSr002, which encodes PUFA synthetase that catalyzes the synthesis of long-chain polyunsaturated fatty acids such as DHA and EPA. In addition, we added chassis endogenous RBS to pfa biosynthetic gene cluster and optimized codon preference to improve the expression efficiency of this module.

Module 3: Suicide Circuit

In the design of the suicide circuit, we innovatively introduced elements from the CRISPRi system and the endogenous toxin-antitoxin (TA) system of the chassis Sinorhizobium fredii CCBAU45436. We bound these components to the inducible promoters in Module 2. This can realize the precise regulation of the system by the external environmental conditions.

In the suicide circuit, vapC encodes the toxin, and vapB encodes the antitoxin. The expression of vapB is regulated by the promoters nifH. Under high oxygen conditions, expression of vapB is suppressed, and under low nitrogen conditions, the CRISPRi system is activated to suppress expression of vapC. When rhizobium escapes from the symbiotic environment and enters a high-nitrogen and high-oxygen external environment, vapC will be expressed normally, while vapB expression will be suppressed by the high-oxygen conditions. This leads to toxin levels exceeding the tolerance threshold of the antitoxin, ultimately triggering the self-destruction of rhizobium. The suicide circuit prevents uncontrolled growth of rhizobium in external environments, ensuring the system's safety and precision.

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