Engineering Escherichia Coli For The Production Of 3-Hydroxypropionic Acid And L-Threonine Using Synthetic Biology Strategies

Synthetic biology plays a-significant role in various fields such as human health,renewable energy,resource utilization,and agricultural protection.The application of synthetic biology strategies in establishing and optimizing biosynthetic pathways for product synthesis,as well as designing and constructing new life systems or organisms with physiological functions,has gained widespread attention in achieving sustainable and green biomanufacturing.Although the high-value chemicals such as 3-hydroxypropionic acid(3-HP)and L-threonine have achieved high production levels,challenges still remain in the production process,including the expression of toxic proteins inhibiting cell growth,insufficient understanding of metabolite transport systems,and unknown cellular physiological metabolic regulation.Therefore,new strategies need to be developed to further enhance the productivity of microbial cell factories,reduce production costs,and achieve global supply-demand balance for these products.Current strategies for creating cell factories through synthetic biology inlude balancing metabolic flux,dynamically regulating gene expression,and synthesizing novel life systems.This paper focuses on Escherichia coli,a commonly used industrial organism with a well-defined genetic background,and explores multiple synthetic biology strategies:(1)fine-tuning the expression of key enzymes,(2)dynamically regulating the expression of transport proteins,(3)dynamically modulating the expression of cell division genes,and(4)constructing polyploid cells.These strategies were then applied to the synthesis of 3-HP and L-threonine,which have high commercial value.1.The expression modulation of the key enzyme Acc for highly efficient 3-HP productionSynthetic biology has created a library of iGEM(International Genetically Engineered Machine Competition)standard biological parts to finely regulate metabolic pathways.In the process of biosynthesizing 3-HP through the malonyl-CoA pathway,acetyl-CoA carboxylase(Acc)plays a crucial rate-limiting step.However,the endogenous Acc in E.coli hinders its efficiency in catalyzing acetyl-CoA to malonyl-CoA due to its toxicity to the cells.In this study,we firstly found that C.glutamicum derived Acc was more conducive to cell growth and 3-HP production.Expressing either subunit of AccBC and DtsRl derived from C.glutamicum Acc individually promoted the growth of E.coli.To further systematically analyze the influence of exogenous Acc on cells,we selected ribosome binding sites(RBS)from the iGEM library with different translational efficiencies to control the expression levels of the two subunits.The results showed that regulating the strength of both subunits promote cell growth and 3-HP production.Additionally,balancing the expression levels of Acc and malonyl-CoA reductase(MCR)is another critical factor for 3-HP production.By adjusting the expression levels of Acc and MCR,we achieved the highest productivity of 3-HP in;E.coli using glucose as the carbon source,reaching 1.03 g/L/h in fed-batch fermentation,along with high titers of 38.13 g/L and a yield of 0.246 g/g glucose.Therefore,precise regulation of the expression of the two subunits AccBC and DtsR1 is an effective strategy to mitigate Acc toxicity to cells.This study established a strain for efficient production of 3-HP through the malonyl-CoA pathway using inexpensive carbon sources.This strategy may also be beneficial for the production of other malonyl-CoA-derived chemical compounds.2.Dynamic regulation of transporter expression to increase L-threonine productionThe dynamic regulation of gene expression using biosensors is a commonly used strategy in synthetic biology for genetic modification.Strong expression of transporter affects the function of other membrane proteins leading to toxicity to the cell.The toxicity of overexpressed transporter limits the chemical production.In this study,we developed a strategy to enhance L-threonine synthesis to dynamically regulate transporter expression.Firstly,we validated that overexpressing the transporter RhtA using constitutive promoters of different strengths increased L-threonine production from 6.51 g/L to 10.02 g/L,but it inhibited cell growth.Then,the effects of dynamic regulation on L-threonine production and cell growth were analyzed by adding IPTG to induce RhtA expression at different periods of cell growth.The results showed that dynamic regulation could further increase L-threonine production to 14.97 g/L,and it was also beneficial to cell growth.To achieve self-induction of transporter,three Lthreonine response activation promoters,PcysJ,PcysD,and PcysJH,were used to regulate the expression of the transporter RhtA,which further increased the L-threonine titer to 21.19 g/L.The feasibility of this dynamic regulatory strategy in enhan cing L-threonine production was also confirmed using other two transporter,RhtB and RhtC This study systematically analyzed the effect of regulating transporter on L-threonine production and developed a novel strategy for dynamic regulation of transporter to enhance L-threonine production.3.Dynamic regulation of E.coli division genes for high-level production of LthreonineRecently,there has been considerable interest in controlling cell division gene expression to produce cell size-dependent chemicals,such as inclusion bodies.In this study,we systematically analyzed the effect of regulating cell division gene expression on the production of conventional chemical L-threonine.First,the effects of the expression of ftsZ,an essential cell division gene,on cells were analyzed using different promoters.The results showed that regulation of ftsZ expression altered cell morphology,growth and physiological metabolism,particularly leading to a 242%increase in fumarate accumulation.Since fumarate is an important precursor for L-threonine synthesis,we hypothesized that dynamic regulation of cell division might promote L-threonine production.Thus,we developed a strong L-threonine biosensor based on the promoter of L-threonine-specific transport protein RhtC.Compared to strains with constitutive ftsZ expression,dynamic regulation of ftsZ was more conducive to Lthreonine production.L-threonine production increased from 21.6 g/L to 37.1 g/L.The accumulation of by-product acetic acid was reduced,from 9.9 g/L to 0.47 g/L.Furthermore,dynamic regulation of division gene expression to promote L-threonine production was also confirmed by other division genes ftsB,ftsL,ftsQ,and zipA.Finally,the engineered E.coli strain produced the highest L-threonine titer of 172.2 g/L for microbial fermentation with a high yield of 1.086 mol/mol and productivity of 2.39 g/L/h in fed-batch fermentation.This strategy might also be developed as a method for.the efficient synthesis of other conventional products.4.Creating polyploid E.coli and its application in efficient L-threonine productionSynthetic biology aims to design and create new living systems.Prokaryotic genomes are generally organized in haploid,but polyploid cells in eukaryotes are more robust.To design and construct a polyploid E.coli,a polyploid E.coli f was successfully designed and created by inserting the weakly expression.part "CmR-Terminator-Promoter-RBS" in front of the chromosome ftsZ gene.PCR amplification showed that cells containing both wild-type and engineered chromosomes.Terminator localization using the green fluorescent protein(GFP)A30 ParB/parS system revealed that cells containing 2,3,or 4 terminators.DAPI staining and flow cytometry analysis showed that 33.2%of cells had 2 chromosomes,57.7%had 3 chromosomes,and 8.9%had 4 chromosomes.Transcriptome analysis showed that the genes of the cell’s main functional pathways were significantly upregulated in the polyploid E.coli,including carbon metabolism,energy metabolism,and amino acid metabolism.Additionally,phenotype analysis revealed increased cell length,enhanced acid tolerance,and improved protein expression performance in polyploid cells.Continuous transfer experiments demonstrated the high stability of ’the polyploid cells’ chromosomes.Finally,we demonstrated that the constructed polyploid cells were beneficial to L-threonine production,achieving the highest reported titer of 159 g/L through fed-batch fermentation.The new biological system constructed in this study provides new substrate for the evolution of prokaryotic cells and indepth studies of polyploid function’s in the future.