L-DOPA Synthesis System Based On Tetrahydrobiopterin Regeneration In Bacillus Licheniformis

Levodopa（L-3,4-dihydroxyphenylalanine,L-DOPA）is a high-value aromatic amino acid derivative existing in animals and plants,which is mainly used for the treatment of Parkinson’s disease.The current production methods of L-DOPA are chemical synthesis,plant extraction and enzymatic catalysis.However,there are many deficiencies,such as environmental pollution,limited sources of raw materials,and the substrate is a Group 2B carcinogen.Therefore,the development of a sustainable recycling L-DOPA synthesis system that meets the food and drug safety level has gradually attracted more attention.This research mainly focuses on the construction of L-DOPA synthesis system using the regeneration of cofactor tetrahydrobiopterin（BH4）in Bacillus licheniformis（B.licheniformis）which is a generally regarded as safe（GRAS）strain.Gene mining techniques and synthetic biology tools were combined with modular metabolic engineering strategies to optimize L-DOPA production of engineered B.licheniformis.The main results are as follows:（1）Rational gene mining methods was first used to screen 7 tyrosine hydroxylases and 6sepiapterin reductases from different microorganisms.Several functional tyrosine hydroxylase/sepiapterin reductase（TH/SPR）synthesis systems were successfully obtained by whole-cell combined transformation method in B.licheniformis.The yield of L-DOPA of Sr TH/Pd SPR synthesis system reached 66.24 mg/L,and the two proteins were derived from Streptosporangium roseum DSM 43021 and Photobacterium damselae subsp.damselae CIP102761,respectively.The production of L-DOPA reached 90 mg/L in the catalysis progress of Sr TH.We found that 7,8-dihydrobiopterin（BH2）,by-product in the catalysis process,has inhibitory effect on Sr TH.It was determined that BH2 formed van der waals force and hydrogen bond with Sr TH and anti-competitive inhibit it in the presence of BH4 through enzymatic kinetics and thermodynamic analysis.The inhibition constant K_i was 98μmol/L.Through adding dihydropteridine reductase（DHPR,EC 1.5.1.34）to the enzymatic system,it was demonstrated that DHPR can alleviate the anticompetitive inhibition of Sr TH by BH2 and promote the synthesis of L-DOPA.（2）The cofactor BH4 regeneration system was constructed on the basis of the screened TH/SPR synthesis system.It was proved that the expression of Pd SPR in B.licheniformis can realize the synthesis of BH4 and the yield of pterin was 8.1 mg/L analzed by secondary mass spectrometry and other methods.The extracellular pterin production was increased to 58.85mg/L after expressing gene fol E2 encoding GTP cyclohydrolase I in recombinant strain R.In order to further enhance the BH4 supply capacity of B.licheniformis,the regeneration of BH4was realized by using the nitroreductase Yfk O derived from B.licheniformis for the first time,which proved that nitroreductase Yfk O has the function of DHPR.It has a good thermal stability and a stronger affinity on substrate 6,7-dihydrobiopterin than other reported DHPR from microorganisms,with a K_m value of 139μmol/L.It was verified that the BH4 regeneration system based on enzyme Yfk O was successfully constructed in B.licheniformis,which can replace the supplementation of cofactor to achieve the same amount of L-DOPA synthesis,with a yield of 93.14 mg/L.However,the continued accumulation of inhibitor BH2 prompted subsequent studies to evaluate this L-DOPA synthesis system by fermentation.（3）The accumulation of precursor L-tyrosine was achieved,and the regulatory mechanism of shikimate pathway was analyzed through expressing and monitoring transcription level of gene.The L-tyrosine production reached 803 mg/L by co-expressing of E.coli-derived genes encoding 3-deoxy-arabino-heptulonate 7-phosphate synthase(Aro G^fbr,D146N)and prephenate dehydrogenase(Tyr A^fbr,M53I/A354V)in B.licheniformis.Other regulated genes aro D and aro K were excavated,encoding shikimate dehydrogenase and shikimate kinase,respectively,by monitoring the transcription level of genes.The L-tyrosine production reached 1200 mg/L after expressing the genes aro D and aro K in recombinant strain HGPA.It was found that the L-tyrosine production reached 1439 mg/L by further supplementing the intermediate shikimic acid.The above proves that the metabolic restriction of the node cannot be completely relieved by gene expression.Through transcriptome sequencing analysis and verification,it was proved that the new transcript 41s RNA plays a regulatory role on genes aro D and aro K in B.licheniformis,and gene yqe I is the"intermediary"in the process of regulation.（4）L-DOPA synthesis system basing on cofactor BH4 regeneration was constructed in B.licheniformis by using modular pathway engineering.Firstly,the BH4 affinity of tyrosine hydroxylase module was improved by site-directed mutagenesis,and the L-DOPA synthesis ability of mutant Sr TH^{V126L F203Y} increased 20.3%.Secondly,the pterin production and NADH/NAD⁺ratio reached 112.48 mg/L and 1.59,respectively,by integrating gene vgb encoding Vitreoscilla hemoglobin（VHb）and deleting gene bud C of competition pathway in BH4 supply module.Then,the precursor L-tyrosine productivity reached 148.0 mg/g _DCW by deleting regulatory factor 41s RNA in L-tyrosine synthesis module.Finally,the BH4regeneration system was integrated into strain 45AB^vCS genome with co-expressing the above three modules to construct recombinant strain 45AB^vCS::PD/HFSRA.The yield of L-DOPA reached 167.14 mg/L and 1290 mg/L,respectively,in shake flask and fed-batch fermentation.The further supplementation of L-tyrosine indicated that the current intracellular supply of L-tyrosine would limit the synthesis of L-DOPA in the fermentation progress of B.licheniformis.