生物基材料组群知识库

羟基脂肪酸是一种重要的化工原料，广泛应用于化学、生物、食品及绿色聚合物材料等行业。近年来，利用生物法制备羟基脂肪酸已经引起了相关研究者的重视，这对解决化学合成所带来的环境污染问题具有重大意义。生物基1,3-丙二醇作为生物基平台化合物，可以用来生产多种高附加值的衍生物，如通过氧化转化反应合成甲酯化产物3-羟基丙酸甲酯、丙二酸二甲酯、丙烯酸甲酯和3-甲氧基丙酸甲酯等，这些甲酯化产物通常是由不可再生的石油或煤炭资源生产。目前，利用可再生的生物质原料替代不可再生的化石资源来制造化学品的研究越来越受到人们的重视。利用1,3-丙二醇和甲醇的氧化转化反应一步法生产甲酯将会成为利用生物基原料生产化学品的一条潜在的可持续的绿色途径。

大肠杆菌作为一种模式微生物，具有遗传背景清晰、易于进行分子操作的优势，工程大肠杆菌已经被广泛用于多种化学品的生产。本研究通过对大肠杆菌代谢通路的改造，分别构建了由葡萄糖和外源脂肪酸生产羟基脂肪酸的工程菌株，为生物法生产羟基脂肪酸奠定了基础。本研究首先通过对大肠杆菌自身脂肪酸合成的调控和外源基因的导入，增加了胞内游离脂肪酸含量。该研究方向主要从以下两个方面入手：（1）改造大肠杆菌自身脂肪酸合成代谢途径，增加由葡萄糖至游离脂肪酸的代谢流；（2）通过敲除fadD基因来切断菌株中游离脂肪酸的降解途径。而后本研究在增加胞内游离脂肪酸基础上，通过异源表达和筛选具有特定活性的脂肪酸羟化酶，并对具有特定链长的羟基脂肪酸的转化条件进行了优化。该研究通过对多个外源基因的组合表达分析，筛选出一株以葡萄糖为底物的羟基脂肪酸生产菌株（敲除fadD基因，共表达P450_BM3和‘TesA基因），该菌株30°C摇瓶发酵15h，羟基脂肪酸产量达到117.0mg/L。同时，为解决细胞内辅酶再生问题并进一步提高产量，本研究还过表达了异源葡萄糖脱氢酶GDH，使羟基脂肪酸的摇瓶产量增加到123.6mg/L。此外，本研究还构建了以外源脂肪酸为底物进行转化反应的菌株（共表达P450_BM3和GDH基因），该菌株在以2.5mM月桂酸为底物时，月桂酸的转化率为96.2%，羟基月桂酸产量为529.0mg/L。但实验发现随着底物中脂肪酸碳链的延长，其羟化效率和产量有降低趋势。

传统的由1,3-丙二醇合成甲酯的工艺须经氧化和酯化两步过程，合成路线长，产物回收率低；而一锅法合成甲酯利用以氧气为氧化剂的醇氧化（1,3-丙二醇氧化成酸）和酯化（酸与甲醇酯化成相应甲酯）过程进行耦合，一锅反应直接得到甲酯化产物。本文在实现形貌可控合成CeO₂载体的基础上，采用沉积-沉淀法合成了3种不同晶型Au/CeO₂催化剂，研究了Au/CeO₂催化1,3-丙二醇氧化转化的反应性能和催化剂结构性质之间的关系。采用XRD、TEM、H₂-TPR、NH₃-TPD和CO₂-TPD等技术表征催化剂的结构和性质，并与其催化性能进行了关联。结果表明，该催化剂具有酸碱催化和氧化催化双功能特性且酸碱中心数量和强度随载体CeO₂的形貌不同而不同。纳米棒和多面体催化剂具有相当数量较强的酸碱性中心，在不添加碱性助剂条件下，Au/CeO₂棒（{110}和{100}）催化剂有利于丙烯酸甲酯的产生（转化率92.3%和选择性41.6%）；以多面体CeO₂（{111}和{100}）为载体的Au/CeO₂催化剂有利于3-甲氧基丙酸甲酯的生成，最大转化率和选择性分别为92.0%和40.2%，首次发现由1,3-丙二醇一锅法合成3-甲氧基丙酸甲酯；以CeO₂立方体（{100}）为载体的Au/CeO₂催化剂有较少的弱酸碱性中心，有利于3-羟基丙酸甲酯和丙二酸二甲酯的合成。Au/CeO₂立方体催化剂能被多次循环利用，且具有良好的再生稳定性。

为增加1,3-丙二醇转化率和产物丙二酸二甲酯的选择性，实验还对立方体载体和催化剂焙烧温度、Au负载量、反应时间和反应压力等影响因素进行了优化，对催化剂酸碱性和还原性与催化性能进行了关联和分析。最后，选择了Pd/CeO₂ 和Au/CeO₂混合催化剂进行研究，相比较单组分催化剂，混合催化剂具有更高的丙二酸二甲酯选择性：1,3丙二醇转化率达到了99.6%，丙二酸二甲酯选择性达到了58.4%，这是目前报道的化学法由1,3-丙二醇一锅法合成丙二酸二甲酯的最高值。

Hydroxy fatty acids (HFAs) are important chemicals with widely application in biodegradable polymer material, chemical, food and mechanical industries. Recently, bioconversion of HFAs is receiving increased interests as the bioprocesses typically use renewable feedstock and do not generate environmental pollution. The bio-based 1,3-propanediol, also known as an important platform chemical for the production of a variety of derivatives, but its methyl esters usually made from non-renewable oil or coal resources. Recently, much attention has been paid to its bio-based production, and to its oxidative transformation into methyl esters in a green way.

As a model microorganism, E. coli possesses the advantage of clear genetic background and is friendly to molecular operations. Engineered E. coli has been widely used in the production of various chemicals. In this study, we reconstructed the metabolic pathway of the host cells and got engineered strains to efficiently produce hydroxy fatty acids from glucose and exogenous fatty acids, respectively. These studies laid the foundation for the large-scale production of hydroxy fatty acids. To modify the native fatty acid synthesis metabolic pathway in E. coli, the heterologous thioesterase genes were engineered to enhance intracellular free fatty acid content; the endogenous fadD gene was knocked out to decrease the fatty acid degradation and thus to enhance the production of FFAs. Once FFAs were accumulated in the biocatalysts, different fatty acid hydroxylases were overexpressed to produce specific chain long hydroxy fatty acids and the biotransformation conditions were optimized. The finally engineered E. coli strain by knocking out the fadD gene and employing the P450_BM3 and 'TesA gave the maximal HFAs production, the hydroxy fatty acid concentration reached 117.0 mg/L under shake-flask conditions. In order to solve the problem of coenzyme NADPH regeneration during reaction, a glucose dehydrogenase (GDH) which is able to regenerate NADP⁺ to NADPH was introduced to further improve the yield. 123.6 mg/L hydroxy fatty acid was finally obtained in shake flask. In addition, an engineered strain was reconstructed to transfer 2.5mM lauric acid as the substrate by coexpressing P450_BM3 and GDH, the conversion rate is 96.2%, and the hydroxy lauric acids reached 529.0 mg/L. As the fatty acid carbon chain extended, the bio-hydroxylation reaction reduced its efficiency.

The conventional two-step route to produce methyl esters from alcohols involves an intermediate step to synthesize the carboxylic acids. However, the processes needed a rather long operation and the product recovery is low, An environmentally benign one-pot route to synthesize methyl esters through an efficient oxidative transformation of alcohols is attracting great interests, which includes oxidation (1,3-propanediol oxidized to acid) and esterification (acid and methanol esterification into corresponding methyl ester). Au/CeO₂ catalysts with different crystal CeO₂ as support were prepared by a deposition-precipitation method and tested for the 1,3-propanediol oxidative transformation. The characterization of the different catalysts was carried out by XRD, TEM, H₂-TPR, NH₃-TPD and CO₂-TPD, and the results were related to the catalytic performance. The results show that the catalytic performance of the Au/CeO₂ catalysts depended on the shapes of the support CeO₂ and the acidic-basic property of Au/CeO₂. These results suggest that the Au/CeO₂ samples are bi-functional catalysts, which possess acid/base catalytic sites and oxidative catalytic site. Their acidic-basic properties could be varied with the CeO₂ shapes. The Au/CeO₂ samples with CeO₂ nanorods and nanopolyhedra as supports contain a certain amount of the stronger acidic and basic sites with broad strength distribution. In the absence of any base, the selectivity of methyl acrylate (41.6% at 92.3% conversion) was greatly improved using the Au/CeO₂ rods ({110} and {100}) as catalysts. To be specific, methyl 3-methoxypropionate was firstly found to be one-pot synthesized from 1,3-propanediol and its maximum conversion and selectivity can be reached to 92.0% and 40.2% respectively by Au/CeO₂ rods ({111} and {100}) catalysts. The Au/CeO₂ cubes ({100}) catalysts with limited weak acidic and basic sites exhibit high selectivity towards methyl 3-hydroxypropionate and dimethyl malonate. Au/CeO₂ cube catalyst can be reused for three cycles without significant deactivation.

To increase 1,3-propanediol conversion and dimethyl malonate selectivity, the influence factors such as calcination temperature of CeO₂ and Au/CeO₂, the pressure of oxygen, reaction time and the Au loading were studied in detail. The preparation conditions were optimized and the results were related to the catalytic performance. Compared with the single catalyst, the Pd/CeO₂ and Au/CeO₂ samples can together catalyze this one-pot reaction with higher dimethyl malonate selectivity: dimethyl malonate selectivity reached 58.4% when 1,3-propanediol conversion reached 99.6%. This was the highest reported dimethyl malonate yield by the 1,3-propanediol oxidative transformation.