Design Of Transcriptional Device Libraries For Precisely Regulated Biosynthesis Of Heterologous Compounds From Methanol In Pichia Pastoris

With the development of theories and methods of synthetic biology,heterologous expression in high-quality chassis hosts has become currently one of the research hotspots and provides a new strategy for the productions of functional proteins and added-value compounds.Most successes have been achieved on model chassis,for example,Escherichia coli and Saccharomyces cerevisiae while there have been less explorations of other non-conventional chassis.Because different chassis have diverse genetic backgrounds,they may possess specific biological components that adapt to particular application scenarios.Therefore,exploration of individualized setups for different chassis hosts is crucial for enriching the theoretical and technical system of synthetic biology and promoting the biosynthesis industrialization.For instance,the bio-utilization of sustainable one-carbon(C1)substrates such as methanol,methane,and CO2 has been attracting wide attention in both academia and industry.The modification of microorganisms that can utilize carbon-substrates as chassis hosts shows great potential for high-efficiency synthesis of valuable products.With mature genetic manipulation tools,some non-methylotrophic chassis,such as E.coli,Corynebacterium glutamicum,and S.cerevisiae,have been introduced with the exogenous methanol assimilation pathway.Nevertheless,their weak methanol utilization ability still lags far behind the production requirements.Instead,natural methylotrophs can efficiently utilize methanol but so far still lack sufficient genetic tools for enabling precise regulation in multigene pathways.The native methylotrophic yeast,Pichia pastoris,has been proven to be an excellent host for protein production and a potentially good chassis for heterologous biosynthesis.This organism can efficiently assimilate methanol and thus grow well on methanol as the sole carbon source.Recently,Golden-Gate,CRISPR-Cas9,and other genome editing strategies have facilitated pathway assembly in P.pastoris.Nevertheless,high-level biosynthesis of the hetelogous compounds requires precise control of the multigene pathway.In comparison to model chassis,P.pastoris still lacks fine-tuned expression tools.In this study,a series of regulation devices with specific functions were assembled based on the design concept of synthetic biology in P.pastoris.Taken together,all the constructed devices constituted a new fine-tuning and intensity-spanned artificial transcriptional regulation device library as powerful tools and platforms for precise regulation in biosynthesis and high production of goal products.In the work,some significant advances have been made.(1)Based on the transcriptional regulation mechanism of the methanol-inducible promoter PAOX1 in P.pastoris,various transcriptional devices were synthesized by fusing three bacterial DNA-binding proteins(DBPs)with three yeast transactivation domains(TFAD)and hybridizing bacterial binding sequences(BSs)with a yeast core promoter.Fine-tuning of the tandem BSs,and nonsense spacers between BS and core PAOX1 further allowed a constitutive transcriptional device library(cTRDL)using the constitutive promoter PGAP as an input promoter.The cTRDL composed of 126 devices with an intensity range of 16%-520%(32.5fold difference)compared with PAOX1(referred to as 100%).(2)By inducible expression,cell growth phase and production phase are separated to release the metabolic burden from recombinant proteins or their catalytic products.Therefore,an inducible transcriptional device library(iTRDL)was further designed for flexible expression control of product synthesis from methanol.A series of methanol-inducible promoters with different activities in P.pastoris were selected as input promoters to assemble iTRDL similar to design of cTRDL.The constructed devices finally constituted a fine-tuning and intensityspanned iTRDL which contains 162 synthetic devices covering an output strength of 30%-500%(16.7-fold difference)compared with the methanol-inducible PAOX1(referred to as 100%).Therefore,a new methanol-responsive expression toolbox was achieved with the excellent regulation performances far more than PAOX1 promoter variants.(3)Monacolin J(MJ),a critical starting material for the semi-synthesis of the commercial hypolipidemic drug simvastatin,was selected as a test model to inspect the applicability of cTRDL and iTRDL for fine-tuning pathway regulation in methylotrophic yeasts.The full pathway of MJ biosynthesis was split into an upstream polyketone synthesis module and a downstream oxidation module.The devices with three expression levels from iTRDL were first picked out to drive four key enzymes involved in the upstream module to separate the cell growth phase and production phase.To precisely balance the DML pathway,an orthogonal design was used with four factors(enzyme-coding genes).Based on data analysis of the orthogonal design,the two enzymes(LovG,LovB)showed significant influence on DML synthesis.A n optimal combination of four enzymes was finally predicted for the balance of synthsis pathway and the production of goal products.Therefore,the optimal strain(Opt)was constructed,and in the shack bottle fermentation of the Opt strain DML titre reached 260.0 mg/L allowing 5.5-fold production compared with the strain of the PAOX1 driving pathway.(4)DML could be oxidized to the target final product of MJ by the downstream module with LovA and its partner CPR.The availability of iTRDL and cTRDL to the downstream pathway of DML to MJ were further investigated in combination with the iTRDL-based upstream pathway module.The devices with three expression levels from cTRDL and iTRDL were selected to drive two key enzymes involved in the downstream module by the full factorial design.As the results,the ingenious balance of the pathways in the downstream module accelerated the conversion of intermediates and improved the production of the target product MJ effectively,and the MJ titre reached 208.0 mg/L(by cTRDL)and 172.0 mg/L(by iTRDL).The highest MJ production from methanol was achieved as more 3.0-fold production as the PAOX1 driving pathway.Consequently,constitutive expression of sLovA and CPR by cTRDL surpassed their inducible expression by iTRDL when adapted to the inducible expression mode of the upstream module by iTRDL.The results illustrated that the strengthen and balance of the pathway nodes inside and between modules could be precisely controlled.We offer new toolboxes and strategies for the high-level biosynthesis of heterologous compounds from methanol in yeast.