Construction And Optimization Of Pichia Pastoris Cell Factories For Vitamin K₂?MK-4? Biosynthesis

Menaquinone(VK2)is a general term for a class of prenylated menadione products with different side chain lengths and belongs to derived lipid.Among them,MK-4 has good effects in promoting blood coagulation and preventing osteoporosis,which has high clinical application value.Escherichia coli and Pichia pastoris,as two attractive expression system that have been successfully applied to the efficient expression of a variety of heterologous proteins.Our research group has successfully achieved the biosynthesis of MK-1 from scratch in E.coli,and the biosynthesis of MK-4 with a longer side chain length will be our next goal.As a eukaryote expression system,P.pastoris has the advantages of rapid growth,easy high-density fermentation,post-translational modification,and easy access to soluble active recombinant proteins.Although the menaquinone biosynthetic pathway has not been discovered in P.pastoris.However,this can also avoid the adverse effects of complex branching pathways and feedback adjustment mechanisms on the pathway reconstruction to some extent.Therefore,this study is based on the concept of synthetic biology and multi-dimensional information integration to achieve efficient biosynthesis of MK-4.We integrate efficient enzymes and biosynthetic pathways into E.coli and P.pastoris to constructe the cell factories that biosynthesizes MK-4 using inexpensive VK1 or VK3 as raw materials,and analyze the advantages and disadvantages of chassis cells to determine the best expression system.The research work not only has effectively expanded the types of chassis cells for VK2 biosynthesis,but also provided a new ideas for the biosynthesis of prenylated products such as VK2,which has great significance for theoretical research and industrial application.Based on the integration of related information from the database,we used BioSynther,a customized biosynthetic potential explorer,to rationally design the MK-4 biosynthetic pathway with VK3 and IPP as substrates.We determined that Homo sapiens UBIAD1(HsUBIAD1)is a key enzyme that catalyzes the polymerization reaction of naphthoquinone skeleton and isopentenyl side-chain.In order to formulate a better protein expression strategy,we carried out relevant bioinformatics analysis on the physicochemical properties,structure and catalytic center of HsUBIAD1.We used the results of bioinformatics analysis to integrate the expression vectors of HsUBIAD1 into the genome of P.pastoris GS115 and combined with multiple screening mechanisms to obtain high-yield recombinant strain GGU-23.Then,the optimum fermentation process parameters of the selected high-yield strain GGU-23 were obtained at the 250 mL shake flask level as follow:initial pH 7.0,24? for 36 h.Under this condition,the biomass of GGU-23 was 31.0 g/L,and the yield of HsUBIAD1 increased by 4.8 times when compared with that under the initial conditions.In order to characterize the enzymatic properties of HsUBIAD1,Ni-NTA affinity chromatography column and gravity desalting column were used to obtain purified HsUBIAD1 protein from recombinant GGU-23 strain,and a preliminary kinetic analysis was conducted.Then,the vitro enzymatic analysis was carried out to determine the optimum enzymatic reaction system and conditions.Under the conditions of initial pH 7.0,31?,and Mg2+ participation,the activity of HsUBIAD1 protein in catalyzing VK3 prenylation was the highest,which was 2.1 times higher than before optimization.The reason is that Mg2+ interacts with key amino acids in the active center of the protein to change its internal conformation,so that the binding ability of the prenyl side chain and the amino acid residues in the active center is enhanced for promoting the enzymatic reaction.In the study whole-cell catalysis,we found that recombinant GGU-23 strain has the ability to catalyze the biosynthesis of MK-4 when using exogenous VKi and VK3 as the isopentenyl acceptor.The content of MK-4 reached 2.03 mg/L when using VK3 as the catalytic substrate.We have achieved a breakthrough in the biosynthesis of MK-4 from the first recombinant P.pastoris strain.In order to obtain a new generation of cell factory,it is necessary to optimize the cell performance of the primary cell factory and improve the biosynthesis of MK-4.We constructed a rDNA-mediated multi-copy integrated expression vector for the expression of SaGGPPS to improve the supply of GGPP,and increased the content of MK-4 in the whole-cell catalytic system by 62.1%.On this basis,the upstream PpIDI was fused with SaGGPPS to further optimize the side-chain biosynthesis pathway,so that the content of MK-4 reached 5.86 mg/L,which was 78.1%higher than before.The new generation of intelligent P.pastoris strain(GGU-GrIG)obtained has achieved the optimization of the biosynthesis capacity of the target product.In order to further explore the application potential of the optimized P.pastoris cell factory,we continue to optimize the whole-cell catalytic process of engineering strain GGU-GrIG.We figured out the optimum process parameters,that is,at 30?,250 rpm,every 6 hours from the beginning of the catalytic reaction,40 mg/L VK3 solution was added to the catalytic system,and continuous cultivation for 18 h was beneficial to MK-4 biosynthesis.After optimization,the yield of MK-4 reached 7.55 mg/L,an increase of 28.8%compared with that before optimization.showing good application potential.The P.pastoris cell factories for MK-4 biosynthesis displayed good application potential.