Metabolic Engineering Of Escherichia Coli To Synthesize Phycocyanobilin

Phycocyanobilin（PCB）is a linear tetrapyrrole compound with unique chemical properties and a wide range of applications in many fields.PCB is safe and healthy as a natural pigment and can be used as a blue additive;as an oral drug with great potential in the treatment of Alzheimer’s disease;as a cosmetic ingredient,it has the functions of anti-oxidation,elimination of free radicals,cell repairment to delay aging,etc.Meanwhile,PCB also has an important role in the field of synthetic biology and is an important component of the optical switch genetic manipulator.At present,the production of PCB mainly relies on the extraction from Spirulina platensis by methanol pyrolysis,but this method has many problems,such as the waste of energy and safety hazards caused by continuous high-temperature pyrolysis,the difficulty of purification due to the diversity of pigments caused by the reaction process,and the long production cycle of Spirulina platensis with production limitations.In addition,the chemical synthesis of PCB has been limited in terms of region-and stereoselective functionalization,and has failed to achieve the synthesis of PCB with high purity and high conversion rate.Studies have shown that PCB biosynthesis can be achieved in microbial systems with the help of genetic and metabolic engineering techniques and means,and this method can effectively reduce production costs,shorten production cycles,and improve product purity,etc.Related research has received increasing attention.In this paper,the de novo synthesis pathway of PCB was constructed using Escherichia coli BL21（DE3）as the original strain,and PCB in fermentation products was identified and quantified;the rate-limiting steps of the PCB synthesis pathway were verified by fermentation strategies such as precursor supplementation and reductant flow addition;efficient synthesis of PCB was achieved by integrating the key genes for precursor substance（heme）synthesis onto the genome of the original strain.In addition,the study also constructed a coenzyme recycling system and an artificial self-assembly complex to enhance the efficiency and yield of PCB synthesis in E.coli;finally,fermentation optimization was performed at the shake flask and 5-L fermenter levels to enhance PCB yield further.The main studies were as follows:（1）Construction of PCB ab initio synthesis pathway in Escherichia coli.By comparing six different E.coli chassis strains,screening two different sources of key enzymes for PCB synthesis and optimizing combinatorial expression,comparing copy number optimization of key enzyme genes for PCB synthesis,assaying fermentation products,and exploring the effect of different copy numbers expression or enhanced expression on PCB synthesis under single cis-trans structure,the synthetic yield of PCB was 8.12 mg/L.（2）Identification of key rate-limiting steps and metabolic regulation of PCB synthesis.To explore the effect of the exogenous addition of 5-ALA and Fe²⁺,as well as some reducing agents,on PCB synthesis and mitigate the rate of degradation in the late stages of PCB synthesis.The key genes for heme synthesis are integrated into the BL21（DE3）genome through CRISPR/Cas9 gene editing and the competitive metabolic pathway for heme synthesis was knocked out.The iron-oxygen reduction-dependent phycocyanin synthase gene pcy A with the iron-oxygen reduction gene fd were fused and expressed by linker to construct a cofactor circulatory system.Ho1 and Pcy A are assembled into artificial self-assembly complexes by the short peptide tag RIAD-RIDD,which promotes the catalytic effect of Ho1 and Pcy A,reduces the synthesis of intermediate product Bv IXα,and improves PCB synthesis,the PCB synthesis of 23.52 mg/L was obtained.（3）Optimization of synthetic process and high-density fermentation to improve PCB production.The best strain was used as the PCB producing strain.Five different fermentation mediums were selected for optimization of the PCB fermentation process,and the effect of induction temperature as well as the concentration of inducer was further explored considering the toxicity of PCB.Finally,the fermentation of PCB production strain was verified and optimized on a 5-L bioreactor.The DO control parameters,inoculum levels,and induction of time points in the fermentation process were compared.The highest PCB yield was reached147.0 mg/L at 36 h.