Static And Dynamic Regulation Strategies Drive (2S)-Naringenin Biosynthesis

(2S)-Naringenin is an important nature product and mainly exists in orange peel,which has lots of physiological functions and is successfully used in food,medicine,and cosmetics fields.Furthermore,as one of the flavonoid scaffolds,（2S）-naringenin can be catalyzed to be most of flavonoid derivatives which have higher additional value.However,the current industrial production method is orange peel extraction,which result low titer,high price and couldn’t meet the market requirements.In this study,static and dynamic regulation strategies and directed evolution strategy were combined to thoroughly regulate（2S）-naringenin synthetic pathway,fatty acid synthetic pathway,and manoly-CoA synthetic pathway.We also established high-throughput metabolic regulation strategy for multi-gene pathway regulation,high-throughput screening methods for（2S）-naringenin production strain screening in multi-well plates and flow cytometry scales,dual-dynamic-regulation system,and multi-system directed evolution theory in this study.Finally,（2S）-naringenin synthetic pathway was rebalanced and dual-dynamic-regulation system was optimized.The theories and achievements in this study paved the way for multi-gene pathway optimization.Major results achieved in this study are highlighted below:（1）Gradient strength promoter-5’UTR complexes screening and engineering in E.coli.Due to the shortage of promoters for metabolic flux fine-tune in E.coli,we screened 104promoter-5’UTR complexes（PUTRs）with gradient strength by RNA-Seq analysis,RegulonDB database analysis,mRNA and protein expression level quantitative analysis.Comparing with 10 mM arabinose induced PUTR_BAD(P_BAD promoter-5’UTR complex),the strength range of candidate PUTRs in transcriptional and translational level are from 0.007%to 4630%and from 0.1%to 137%,respectively.Furthermore,to further improve PUTR strength,we combined different promoter and 5’UTR regions together or directly cascade PUTRs with different translational and transcriptional levels together to construct to construct a series of combinational PUTRs or cascade PUTRs.Finally,two combinational PUTRs(P_ssrA-UTR_rpsT and P_dnaKJ-UTR_rpsT)and cascade PUTRs(PUTR_ssrA-PUTR_infC-rplT and PUTR_alsRBACE-PUTR_infC-rplT)have highest strength with 170%,137%,409%,and 203%of that of 10 mM arabinose induced PUTR_BAD,respectively.（2）Establishing diverse scale of high-throughput screening methods for（2S）-naringenin production strain screening.Metablic engineering usually be combined with directed evolution to faster achieve the goals.However,the only method for（2S）-naringenin quantity is high performance liquid chromatography（HPLC）,which can not achieve the goal of high-throughput.Based on the reaction theory between metal ion（AlCl₃ and Mg（Ac）₂）and（2S）-naringenin,we established and optimized the multi-well plate based UV spectroscopy（373 nm）and fluorescent spectrometry（382 nm/505 nm and 371 nm/492 nm）high-throughput screening methods.On the other hand,flow cytometry based screening method was also established by using（2S）-naringenin concentration responded protein TtgR.Combining with far red fluorescence protein（FRFP）,the fluctuate of plasmid copy number and cell size between different single cells were nomilized.Hence,combining the two high-throughput screening method together results a 10³-10⁷/d screening throughput,which can cover most of metabolic engineering requirements.（3）（2S）-Naringenin synthetic pathway optimization by iteration high-throughput screening strategy.It is difficult to regulate all genes with different expression level in multi-gene pathway at the same time.To overcome this challenge,we established a series of PUTR libraries with different expression scale by using screened gradient strength PUTRs.The promoter libraries were then randomly coloned into the upstream of（2S）-naringenin synthetic pathway genes.Finally,we built a randomly optimized plasmid library.The construction efficiency and PUTRs emergency rate of all of the libraries were higher than 83%and 60%,respectively.Then the best strain was obtained by iteration high-throughput screening strategy in 96-well plates.The highest（2S）-naringenin titer is 192 mg·L^-1,and p-coumaric acid accumulation is 29 mg·L^-1.In addition,different with the previous reports,we find CHS is the rate-limiting step in our system.（4）Enhancing malonyl-CoA production by dual-dynamic-regulation system.Based on lots of previous reports,malonyl-CoA shortage was considered as one of the mainly bottleneck for（2S）-naringenin production.Malonyl-CoA consumption and synthesis dynamic regulation systems were established by using（2S）-naringenin and p-coumaric acid responded protein FdeR and PadR,respectively.The cell growth and（2S）-naringenin synthesis were separated into two stages based on（2S）-naringenin and p-coumaric acid production.Furthermore,fatty acid synthetic pathway and malonyl-CoA synthetic pathway were further optimized and resulted in 7.4-fold improvement of malonyl-CoA production.Finally,comparing with the strain without dynamic regulation system,（2S）-naringenin titer was improved by 3.2-and 1.5-fold,respectively,by using malonyl-CoA consumption and synthesis dynamic regulation systems.Combining the two dynamic regulation systems into one strain resulted in（2S）-naringenin 3.8-fold improvement and reached 139 mg·L^-1.（5）Coupling and optimization of dual-dynamic-regulation system by directed evolution strategy.The multiple artificial regulation systems usually couldn’t couple with each other in the same host.To couple with the constructed dual-dynamic regulation systems,we used directed evolution strategy to regulate the expression level of FdeR and PadR.After error-prone PCR and high-throughput screening process by flow cytometry,the selected strain coupled dual-dynamic regulation systems and has the highest（2S）-naringenin production.Finally,the（2S）-naringenin titer reached 251 mg·L^-1,which is 1.8-fold of none directed evolution strain.Then fermentation temperature,initial pH,carbon and nitrogen sources were optimized.After the fed-batch fermentation in flask and biolector microreactor,（2S）-naringenin titer reached 282 mg·L^-1.