以碱性纤维素溶液为基质生产可降解地膜用P3HB4HB材料研究

In this study, polyhydroxybutyrates producing pathways will be reconstructed in Micrococcus luteus R17 aiming at increasing the substrate to PHA yield and utilizing low cost carbon sources：such as cellulose for production of copolymers of 3-hydroxybutyrate (3HB) and 4-hydroxybutyrate (4HB). .NAD kinase will be over expressed in Micrococcus luteus R17 to enhance the accumulation of poly-3-hydroxybutyrate (P3HB). The recombinant Micrococcus luteus R17 harboring P3HB synthesis pathway was engineered to have an accelerated supply of NADPH, which is one of the most crucial factors influencing P3HB production. Excess NAD kinase in Micrococcus luteus R17 harboring P3HB synthesis operon phbCAB increased the accumulation of P3HB and yields of substrate to P3HB. .A metabolically engineered Micrococcus luteus R17 will be constructed for the production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P3HB4HB) from cellulose carbon sources. Genes involved in succinate degradation in Clostridium kluyveri and P(3HB) accumulation pathway of Ralstonia eutropha were co-expressed for the synthesis of the above copolyester. Micrococcus luteus R17 native succinate semialdehyde dehydrogenase genes sad and gabD were both deleted for eliminating succinate formation from succinate semialdehyde, which functioned to enhance the carbon flux to 4HB biosynthesis..Based on the recombinant Micrococcus luteus R17, a sterilization free and continuous fermentation process will be developed, which provide a new solution for reducing cost of P3HB4HB production.

为解决生物降解地膜成本高居不下，“推而不广”等问题，本课题以NaOH/Urea-纤维素取代葡萄糖底物，通过在藤黄八叠微球菌Micrococcus luteus R17中异源表达罗氏真养杆菌的P3HB合成操纵子phaCAB和克氏梭菌的琥珀酸降解相关基因sucD、4hbD和orfZ，并对菌株琥珀酸半缩醛脱氢酶基因sad和gabD进行敲除，构建能够利用纤维素作为碳源来合成3-羟基丁酸(3HB)和4-羟基丁酸(4HB)共聚酯（P3HB4HB）的工程菌。利用野生菌株胞内NAD激酶的过表达，提高胞内辅酶NADP(H)的水平，提高碳源到产品的转化率。围绕重组Micrococcus luteus R17菌株，开发配套无灭菌、连续发酵、流加纤维素碱溶液为培养底物的发酵工艺，并进行吹塑与可降解地膜试制。为降低可降解地膜用P3HB4HB材料生产成本提供新的解决方案。

P3HB4HB是PHA家族中一类具有优良材料学性能的高聚物材料，是制造环境友好性地膜的理想材料，制约其大规模应用的主要制约是高居不下的生产成本。如能将农业产生的大量植物秸秆纤维素溶解于碱性溶剂，以此作为发酵底物，可降解纤维素的细菌为底盘生物，建立发酵体系生产低成本P3HB4HB，将P3HB4HB用于农用地膜等各种包装材料，地膜可通过微生物分解直接回到农田环境，从而实现绿色循环。亟待解决的科学问题是：1、开发快速、廉价的纤维素溶解方法及相关培养基质，筛选可耐受该碱性溶液专用菌株；2、对此菌株进行从纤维素到P3HB4HB的合成途径改造；3、开发与上述基质、菌株相配套的发酵方法。针对上述问题，本课题以农业生产产生的大量废弃纤维素为底物，因其能够在添加尿素的氢氧化钠溶液里大量溶解，通过优化组分配比，开发出一种可快速溶解秸秆纤维素、有效屏蔽环境杂菌、可回收使用的NaOH/Urea-纤维素基质。.筛选出可在NaOH/Urea-纤维素基质中生长并有效将纤维素进行转化的藤黄八叠微球菌菌株。测序并绘制了Micrococcus luteus R17全基因组完成图（Gene Bank Accession: PRJNA418074 ID: 418074）。通过敲除fadB2x及fadAx，将Micrococcus luteus R17的β氧化途径阻断，构建突变株；用混合碳源调控P3HB4HB单体组成比例的可行性。在野生菌Micrococcus luteus R17的P3HB4HB生物合成途径中，来自脂肪酸β-氧化的中间体烯脂酰辅酶A在烯脂酰辅酶A水合酶（PhaJ）的催化下，水合形成P3HB4HB的两种前体3HB-CoA和P3HB-CoA，然后在P3HB4HB合酶（PhaC）的催化下聚合为P3HB4HB。结合碱液前处理秸秆纤维素机制的研究，以纤维素的碱性水溶液为重组Micrococcus luteus R17菌株的发酵底物，开发出一种新的无灭菌、连续发酵、流加纤维素碱溶液的方法来生产P3HB4HB。.发表论文5篇，其中SCI论文2篇，影响因子均大于5；制作计算机软件“基于Unity3D的可降解塑料生产过程的设计与实现”取得著作版权。