| Medium-chain dicarboxylic acids?MCDs?are important biochemical platform compounds,which are widely used in organic synthesis,food flavoring,medical and other fields.In addition,MCDs are also key monomers of nylon polymers.Since the variety of superior properties,nylon materials are widely used in machinery,chemical,and medical fields.However,the current industrial production of MCDs mainly relies on chemical synthesis,which leads to excessive emission of nitrogen oxides.Therefore,it is important to establish an alternative approach based on microbial fermentation to achieve the cleaner production of MCDs.Although MCDs such as adipic acid,glutaric acid,pimelic acid,and suberic acid were synthesized by fermentation approaches,the large-scale production was limited due to the low titer and yield.In this study,we focused on the heterologous biosynthesis of MCDs in Escherichia coli to demonstrate the feasibility of using metabolic engineering and synthetic biotechnology to produce MCDs.Through a series of strategies such as pathway de novo design,multivariate modular metabolic engineering,suppression of competitive pathways,increase of precursors,and fermentation optimization,the highly efficient biosynthesis of adipic acid and glutaric acid were achieved in E.coli.The main results were described as follows:?1?De novo biosynthesis of adipic acid by reverse adipate-degradation pathway?RADP?.Firstly,the Thermobifida fusca RADP was reconstructed in E.coli BL21?DE3?for adipic acid production.The RADP included Tfu?0875??-ketothiolase?,Tfu?2399?3-hydroxyacyl-Co A dehydrogenase?,Tfu?0067?3-Hydroxyadipyl-Co A dehydrogenase?,Tfu?1647?5-carboxy-2-pentenoyl-Co A reductase?and Tfu?2576-7?succinyl-Co A synthase?.Then,preliminary optimization of fermentation conditions,the adipic acid was produced when the OD600 reached 0.80 and adding 1 m M isopropyl-?-D-thiogalactoside?IPTG?in SOB medium.The fermentation results of our recombinant strains Mad136 and Mad236 showed that the high-intensity expression of Tfu?0875 and Tfu?2399 was more conducive for producing adipic acid.Finally,4.77 g·L-1 adipic acid was successfully obtained by fermentation of Mad136 under 1 vvm aeration.?2?Optimization of the expression of adipate acid biosynthetic metabolic network.In order to further enhance the metabolic flux and supply precursors for the RADP pathway,the gene sources and expression levels of the RADP were optimized,and the rate-limiting steps were analyzed.When different promoters regulated the expression of Tfu?1647,it is found that the expression effect of Trc promoter is better than T7 promoter.Comparing the different sourced genes,we found that the RADP derived from T.fusca has higher adipate synthetic efficiency.Furthermore,we optimized the by-pass pathways by knocking out the lactate dehydrogenase?Ldh A?,succinyl-Co A ligase?subunit?Suc D?,and acetoacetyl-Co A transferase?Ato B?to re-direct the cofactor and carbon flux into the biosynthesis of adipic acid.Finally,the best strain Mad123146 was sub-cultured by 10-rounds of continuously fermentation in shake flasks in order to test its genetic stability.And the production of adipic acid was maintained above 1.50 g·L-1,which confirmed its high genetic stability.?3?Optimization of the fermentation conditions and exploration of purification process for adipic acid.The dissolved oxygen and fed-batch fermentation conditions were optimized for the best strain Mad123146 and 68.00 g·L-1 adipic acid was successfully obtained in E.coli.Then,according to the physical and chemical properties of adipic acid,the purification process of adipic acid in the fermentation broth was established through ethyl acetate extraction,membrane filtration,rotary evaporation,and semi-preparative liquid phase separation.?4?De novo biosynthesized of glutaric acid by RADP.Tfu?0875 of RADP have a broad substrate spectrum.According to the Gas-phase mass spectrometry?GC-MS?and liquid-phase mass spectrometry?LC-MS?results,we firstly confirmed that glutaric acid also could be synthesized by RADP.Then,the biosynthetic metabolic network,fermentation medium,dissolved oxygen,induction concentration,and the inhibitor addition were optimized to improve the production of glutaric acid.And 4.82 g·L-1 glutaric acid was obtained in 5-L reaction,which was 5358-folds higher than the starting strain Bgl146.In order to further enhance the supply of glutaric acid precursor malonyl-Co A and acetyl-Co A,acetyl-Co A carboxylase?ACC?and acetyl-Co A synthase?acs?were over-expressed and the glutaric acid production finally was increased to 7.97 g·L-1 in the 5-L reaction.?5?Construction of a high gradient strength constitutive promoter library.In order to avoid the addition of IPTG in industrial MCDs production process,the constitutive promoters were required to optimize the expression of RADP.However,the strength of the existing constitutive promoters couldn't satisfy the requirements of pathway expression.In order to obtain high gradient strength constitutive promoters,a de novo mutant promoter library was constructedusingtrcpromoter(Ptrc)asatemplateby mutation-construction-screening-characterization?MCSC?engineering cycles.After Mi Seq of the mutant promoter library,mutations mainly occurred in the core and downstream regions.Based on the above-mentioned mutation,a synthetic promoter library was constructed.The maximum strength of the promoter in this library was about 69-folds that of Ptrc,and the strength span between the strongest and weakest promoter is about 454-folds.The expression strength of these synthetic promoters exhibited a high correlation on either plasmid or genome when expression?-galactosidase?Lac Z?,lactate dehydrogenase?Ldh A?,and sf GFP.In order to accurately prediction of promoter strength,a model of the relationship between promoter sequence and strength was established based on different machine learning models.Finally,we found that Xg Boost was the best model?R2=0.77?.This study can provide high gradient strength promoter elements for the constitutive synthesis of MCDs without inducer in E.coli. |
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