代谢工程可以定向的改造微生物高效生产目的产物。聚羟基脂肪酸酯（PHA）家族是一类由微生物合成的具有生物相容性和生物可降解性的高分子聚合物，其种类众多，根据组成的单体以及空间排列结构不同，表现出不同的理化性质。目前PHA的生产应用主要面临两大问题，高成本和低附加值，聚3-羟基丙酸酯是一种具有高强机械性能的新型PHA材料，受3-羟基丙酸单体的影响，其共聚物也会表现出优异的性能。本论文设计新型代谢途径，以大肠杆菌为宿主，生产具有高附加值的含有3-羟基丙酸单体的生物聚合物。以葡萄糖为碳源生产具有高附加值的聚3-羟基丙酸酯（P3HP）以及3-羟基丙酸和3-羟基丁酸的共聚物。来源于酿酒酵母的基因gpd1和gpp2分别编码3-磷酸甘油脱氢酶和3-磷酸甘油磷酸酶，可以催化糖酵解的中间产物磷酸二羟丙酮生成甘油，来源于克雷伯氏菌的基因dhaB123编码甘油脱水酶，并在基因gdrAB编码的甘油脱水酶激活酶的帮助下，可以利用甘油产生3-羟基丙醛，然后被来源于鼠伤寒沙门氏菌的基因pduP编码的丙醛脱氢酶代谢为3-羟基丙酰辅酶A，最后利用罗氏真养菌来源的PHA聚合酶生产聚3-羟基丙酸酯，在LB培养基中，重组大肠杆菌细胞干重可达5.35 g/L，P3HP占细胞干重的百分含量为18.4%，并在此基础上添加经典的P3HB合成基因phaA和phaB，在11个外源基因组成的代谢通路作用下，大肠杆菌可以利用葡萄糖合成3-羟基丙酸和3-羟基丁酸的共聚物P3HB3HP。3-羟基丙酸和4-羟基丁酸的共聚酯（P3HP4HB）是一种仍未在天然微生物体内报道合成的聚合物，通过代谢工程的手段构建了一条由五个外源基因组成的代谢通路，包括来源于恶臭假单胞菌KT2442的基因dhaT和基因aldD，分别编码1,3-丙二醇脱氢酶和醛脱氢酶，来源于橙色绿屈挠菌的基因pcs’编码丙酰辅酶A连接酶，来源于克氏梭菌的基因orfZ编码4-羟基丁酰辅酶A转移酶，来源于罗氏真养菌的基因phaC编码PHA合成酶，使大肠杆菌可以利用1,3-丙二醇和1,4-丁二醇生产P3HP4HB，并且通过碳源比例的调节，3HP单体的摩尔比例可从18%调节到88%，PHA的热力学性能和透明度都会随着单体比例的变化而变化。本论文的工作利用代谢工程的方法构建了多条代谢通路，合成出新型的PHA材料，为高附加值PHA生物材料的研究和应用打下了理论基础。
Metabolic engineering has been exploited as a powerful approach for enhanced production of chemicals and biopolymers. Polyhydroxyalkanoates (PHA) are a family of biocompatible and biodegradable polyesters produced by various microorganisms. PHA exhibit diverse properties depending on the monomer structure and composition. PHA are still facing these problems of high cost or poor properties. The homopolymer poly(3-hydroxypropionate) (P3HP) is the strongest family member of microbial PHA, and the 3-HP containing copolymer also shows good properties. This study focused on metabolic engineering of Escherichia coli to synthesize 3-HP containing PHA polymers for possible high-value applications.Synthesis pathways of P3HP and its copolymer P3HB3HP of 3-hydroxybutyrate (3HB) and 3-hydroxypropionate (3HP) were assembled respectively to allow their synthesis from glucose, a more abundant carbon source. Recombinant Escherichia coli harboring the P3HP synthetic pathway consisting of heterologous genes encoding glycerol-3-phosphate dehydrogenase (gpd1), glycerol-3-P phosphatase (gpp2) from Saccharomyces cerevisiae that catalyze formation of glycerol from glucose, and genes coding glycerol dehydratase (dhaB123) with its reactivating factors (gdrAB) from Klebsiella pneumoniae that transfer glycerol to 3-hydroxypropionaldehyde, as well as gene encoding propionaldehyde dehydrogenase (pduP) from Salmonella typhimurium which converts 3-hydroxypropionaldehyde to 3-hydroxypropionyl-CoA, together with the gene of PHA synthase (phaC) from Ralstonia eutropha which polymerizes 3-hydroxypropionyl-CoA into P3HP. The recombinant E. coli grown on glucose LB medium successfully produced 5.35 g/L cell dry weight containing 18.4% P3HP.When phaA and phaB from Ralstonia eutropha respectively encoding β-ketothiolase and acetoacetate reductase, were introduced into the above P3HP producing recombinant E. coli, copolymers P3HB3HP were synthesized from glucose as a sole carbon source.Poly(3-hydroxypropionate-co-4-hydroxybutyrate) was produced by E. coli harboring a synthetic pathway containing five heterologous genes including dhaT and aldD encoding 1,3-propanediol dehydrogenase and aldehyde dehydrogenase from Pseudomonas putida KT2442, pcs’ encoding propionyl-CoA ligase from Chloroflexus aurantiacus, orfZ encoding 4-hydroxybutyrate coenzyme A transferase from Clostridium kluyveri and phaC encoding PHA synthase from Ralstonia eutropha. Compositions of 3HP in microbial P(3HP-co-4HB) were controllable ranging from 18 mol% to 88 mol% depending on 1,3-propanediol/1,4-butanediol ratios. The thermal and mechanical properties, transparency also showed obvious changes depending on the monomer ratios. In this study, various 3HP-containing polymers were synthesized and the production of P3HP from glucose allows the strong P3HP to become more attractive in term of the production cost.
