Alginate production by mutants of Azotobacter vinelandii: advanced strategies for cultivation under microaerophilic conditions and overpressure

高粘性发酵液是生物工艺开发和结垢过程中经常遇到的问题之一。多糖的生产可以看作是这种过程的一个例子。在微生物多糖领域，海藻酸盐是最重要的一类。褐藻酸盐是一种由- d -甘露醛酸(M)和- l -古鲁醛酸(G)组成的聚合物，由褐藻和固氮杆菌、铜绿假单胞菌等细菌产生。海藻酸盐具有广泛的应用范围，因为它们可以作为粘稠剂和凝胶形成剂，使其成为许多工业领域的极大兴趣。由于大多数海藻酸盐生物合成的细节是已知的，以及这一途径与A. vinelandii中存在的其他途径的代谢关系，因此有可能产生这种生物聚合物的过量菌株。此外，基因修饰允许影响生产的海藻酸盐的物理和化学性质。此外，先前的研究表明，在非常低的溶解氧张力（微嗜气条件）下，可以操纵A. vinelandii产生的海藻酸盐的分子质量和乙酰化程度。因此，本提案的最高优先级是研究微嗜氧条件的潜力，以显着最大化转基因菌株形成具有特定化学特性的藻酸盐。海藻酸盐的形成导致发酵液的非牛顿流体行为，对混合均匀性、液体输入功率、热量去除以及总体上对结果的再现产生重大影响。因此，从摇瓶到搅拌槽反应器的规模化是该生物系统面临的主要挑战。因此，在摇瓶水平、实验室规模的搅拌槽反应器以及50 L中试规模的压力反应器上进行工艺放大和结果再现也将是本提案的重要组成部分。此外，该项目还将为学生和科学家提供在墨西哥国立墨西哥大学生物技术研究所Peña博士的墨西哥小组和亚琛工业大学Regestein博士的德国小组进行研究的机会。

Highly viscous fermentation broths are one of the problems frequently encountered in the development and scaling of bioprocesses. The production of polysaccharides can be considered as an example of such processes. In the field of microbial polysaccharides, alginates are counted among the most important ones. Alginate is a polymer composed of beta-D-mannuronic acid (M) and alpha-L-guluronic (G) acid and produced by brown algae and bacterial species such as Azotobacter vinelandii and Pseudomonas aeruginosa. Alginates have a wide scope of application due to their use as viscosifiers and gel forming agents, making them of great interest to several industrial areas. Because most details of the alginate biosynthesis are known, as well as the metabolic relationships of this pathway with other pathways present in A. vinelandii, it was possible to generate overproducing strains of this biopolymer. Moreover, genetic modifications allow to influence the physical and chemical properties of the produced alginate. In addition, previous studies have shown that for very low dissolved oxygen tensions (microaerophilic conditions), it is possible to manipulate both the molecular mass as well as the degree of acetylation of alginate produced by A. vinelandii. For this reason the highest priority of this proposal is to investigate the potential of microaerophilic conditions to significantly maximize the alginate formation with specific chemical characteristics by genetically modified strains of A. vinelandii. The formation of alginate leads to a non-Newtonian fluid behavior of the fermentation broth with a significant impact on mixing homogeneity, power input into the liquid, heat removal and in general on the reproduction of results. Thus, the scale up from shake flask to stirred tank reactor is a major challenge for this biological system. Process scale up and reproduction of results on shake flask level, lab scale stirred tank reactor as well as in a 50 L pilot scale pressure reactor will therefore also be an important part of this proposal. In addition, this project will give students and scientists the opportunity for research stays in the Mexican group of Dr. Peña at UNAM Institute of Biotechnology and the German group of Dr. Regestein at RWTH Aachen University.