Metabolic Engineering Of Bacillus Subtilis For Efficient Synthesis Of Menaquinone-7

Menaquinone-7?MK-7?is one of fat-soluble vitamin that plays an important role in preventing cardiovascular sclerosis and treating osteoporosis.MK-7 has attracted much attention in the fields of medicine and functional food due to its advantages of long half-life period and high biological affinity.At present,MK-7 is mainly obtained via fermenting natto with Bacillus subtilis natto.The fermentation process,however,has limitations such as low biosynthesis efficiency,long fermentation cycle,and difficulties in extraction.Therefore,it is urgent to obtain a recombinant strain that synthesizes MK-7 efficiently by enhancing the synthesis pathway of MK-7 and exploring the factors that limit the biosynthesis of MK-7.In this thesis,the food-safe strain Bacillus subtilis 168 was determined as the starting strain.Meanwhile,methods like metabolic engineering technology,synthetic biology,and transcriptomics were adopted to enhance precursor supply,construct population response regulatory elements,and stabilize electron transfer.At last,a strain with high MK-7 titer was obtained.The main research points of this thesis are as follows:?1?In B.subtilis 168,the native promoter in the men gene cluster was replaced by a constitutive promoter to eliminate the influence of promoter regulation on the synthesis of MK-7.Then the constitutive promoter was used to drive PEP synthase?Pps A?,transketolase?Tkt?,shikimate kinase?Aro K?,1-deoxyxylulose-5-phosphate synthase?Dxs?,1-deoxy-D-xylulose-5-phosphate reductoisomerase?Dxr?and other coding genes resulting the accumulation of chorismite and isoprene pyrophosphate,then,added the copy number of 1,4-dihydroxy-2-naphthoate octadienyl transferase gene men A in the genome.The MK-7 titer of the recombinant strain in a 250 m L shake flask was 75 mg/L,about 8.33 times than that of the original strain.?2?In addition to truncating the N-terminus of histidine kinase Kin A and expressing it by constitutive promoter,we also knocked out the early genes spoii A and spoii E to ensure the Spo0A continually phosphorylated and reduce the formation of spores.Then we used degenerate primers to mutate the Spo0A-P binding site and obtained a binding site"ATTGACAGGGAA"with stronger affinity.Subsequently,activation and repressor promoters with different expression intensity were obtained by changing the sequence and number of spo0A-P binding sites on promoter P_spoiia and P_abrb.Finally,we clarified the relationship between the signal molecule Phr60 and the regulator Rap60 via the experiment of changing copy numbers.Besides,we also figured out the relationship between Phr60 and cell growth.After establishing the model between cell density and promoter,we constructed the Phr60-Rap60-Spo0A population response regulation system.?3?To achieve a dynamic balance between the synthesis of MK-7 and cell growth,we worked in two parts.In the first place,the activated promoter P_{spoiia?cs-1,3?}was used to dynamically drive the genes isp H,hep S/T,and the key genes of the toxic metabolites synthesis to force the toxic metabolites flux to HDP as soon as possible.Secondly,the native promoters of pyk and upp S encoding genes were replaced in situ by the repressible promoter P_{abr B?cs-1?},inhibiting the genes related to cell growth,in case making the carbon flux to the target product synthesis way.After six days of fermentation in the shake flask,the titer of MK-7 reached360mg/L.In the meantime,the MK-7 specific synthesis rate and productivity in BS20 was 51.5mg/?L·g DCW?and 2.5 mg/?L·h?,respectively.The titer of MK-7 reached above 200 mg/L in5-L,15-L and 3-T bioreactors and the fermentation cycle was significantly shortened.?4?In order to clearly clarify the restricting factors of MK-7 synthesis.Firstly,the transcriptional level of B.subtilis 168 genes between shaking culture?no biofilm formation?and static culture?biofilm formation?was analyzed via transcriptomics.In result,the differential genes were related to membrane components,such as transmembrane transporters?BSU29340,BSU03070?.Then the expression of oxalate decarboxylase Oxd C has been observed very abundant during the static culture of B.subtilis 168 via SDS-PAGE and protein flight mass spectrometry.The Oxd C can provide a large number of electrons for the respiratory chain.Thus,we combinatorically overexpressed the cell membrane protein Tat AD-CD and the cytochrome C reductase Qcr A-C in the strain BS20,obtaining a recombinant strain BS20-QT.As a result,the MK-7 titer,was significantly increased from 200 mg/L to 310 mg/L in the 15 L fermenter,with the specific synthesis rate and the productivity of MK-7 reaching 22.14mg/?L·g DCW?and 3.22 mg/?L·h?,respectively.