Engineering Of Xylose Nonphorsphorylative Metabolism And Shikimate Pathway For The Biosynthesis Of Value-added Products

With the depletion of petroleum resource and the deterioration of the natural environment,utilization of renewable carbon sources to construct microbial cell factory for the synthesis of chemicals,fuels and pharmaceuticals has becoming more and more important.With the rapid development of metabolic engineering and synthetic biology,many natural products have been achieved biosynthesis.However,due to the lack of known metabolic pathways or related enzymes,biosynthesis of a lot of non-natural and high-value compounds have yet been achieved.In order to overcome this issue,we used enzyme promiscuity and protein engineering strategy to expand the substrate spectrum of the known enzymes,then to construct artificial biosynthetic pathways to produce six kind of high value compounds.Lignocellulose is the most abundant and cheap feedstock on the earth.Xylose as the second most abundant sugar in lignocellulose,its catalytic efficiency in microorganisms is lower than glucose.Thus,it is quite important to develop novel metabolism and optimization strategies for converting xylose into target products.In this study,we constructed and engineered xylose nonphosphorylative metabolic platform,which is short,efficient and has higher theoretical molar yield compared with the xylose traditional metabolic pathway.Based on this,we constructed a novel biosynthetic pathway towards 3,4-dihydroxybutyric acid.Screening of the key pathway enzymes,identification of a novel 3,4-dihydroxybutanal dehydrogenase and disruption of several competing pathways enabled the production of 1.27 g/L 3,4-DHBA in shake flask experiments,which is the highest titer reported so far.Then we rationally engineered a diol dehydratase to achieve non-native catalysis of 1.2,4-BTO into 1,4-BDO by releasing the substrate inhibition effect,enlarging the catalytic pocket and promoting the maintenance of O1-M ion distance.Then we optimized the 1,2,4-BTO biosynthesis from xylose by disruption of the competing pathways.When introducing the best diol dehydratase mutant into the 1,2,4-BTO producer,the engineered strain generated 209 mg/L 1,4-BDO from xylose in shake flasks.Finally,we investigated the maximum production potential of different xylose metabolic pathways and illustrated for the first time that xylose isomerase pathway and Weimberg pathway are the synergetic pathways for the production of acetyl-CoA and ?-ketoglutarate derived products.When using glutarate as the target product,the combination of those two synergetic pathways led to 602 mg/L glutarate,which is higher than using each single pathway alone(104 or 209 mg/L),even higher than using glucose as the sole carbon source for synthesis of glutarate(420 mg/L).Our work not only constructed a novel and efficient xylose metabolism for the synthesis of value-added products,but also demonstrated a novel xylose efficient utilization strategy.Phenolic compounds are a member of aromatic compounds.They have attracted much attention due to their outstanding bioactivities,such as antibacterial,anti-inflammatory,antioxidant,anti-cancer,anti-aging,anti-UV and antiseptic properties.However,the content of these compounds in nature is very low,thus limiting their large-scale commercial application.In this study,we achieved biological synthesis of three important phenolic compounds(pyrogallol,salicyl alcohol and gentisyl alcohol)for the first time by engineering the shikimate pathway.First,we discovered for the first time that a small protein PHQ present in the genome of Pseudomonas stutzeri OX1 promotes the catalytic activity of phenol hydroxylase PH.We then explored the substrate spectrum of PH and found that it can catalyze some non-natural substrates such as catechol,4-hydroxy benzoic acid and resorcinol.On this basis,we coupled the biosynthetic pathway of catechol with the PH-catalyzed hydroxylation reaction and constructed a new pathway for the production of pyrogallol.Then,we constructed another biosynthetic pathway towards pyrogallol by recruiting salicylate 1-monooxygenase.We identified and characterized a highly efficient 2,3-dihydroxybenzoic acid monooxygenase from a series of oxygenases and hydroxylases based on the structural similarity of substrates and products.Expression of 2,3-dihydroxybenzoic acid synthase and 1-monooxygenase,enhancement of the carbon flux into the shikimate pathway,modular optimization of the pathway enzymes and alleviation of the autoxidation of the product enabled the production of 1035.75 mg/L pyrogallol in shake flask experiments.Finally,we achieved biosynthesis of salicyl alcohol and gentisyl alcohol from renewable sources for the first time.By using a broad substrate spectrum carboxylic acid reductase CAR,we successfully realized the synthesis of salicyl alcohol and gentisyl alcohol from salicylate and gentisate respectively.Then we used a new salicylic acid-5-hydroxylase enables synthesis of gentisate from salicylic acid.Finally,we constructed de novo synthesis pathways of salicyl alcohol and gentisyl alcohol.Optimization of the carbon flux through the shikimate pathway enabled production of 594.4 mg/L salicyl alcohol and 30.1 mg/L gentisyl alcohol.Our work not only achieved biosynthesis of several phenolic compounds for the first time,but also demonstrated utilization of enzyme promiscuity to construct non-natural biosynthetic pathways to produce value-added products.