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

高粘度发酵液是生物过程开发和规模化过程中经常遇到的问题之一。多糖的生产可以被认为是这种过程的一个例子。在微生物多糖领域，藻酸盐是最重要的一类。海藻酸盐是由褐藻和棕色固氮菌、铜绿假单胞菌等细菌产生的一种由β-D-甘露糖酸(M)和α-L-古糖醛(G)酸组成的聚合物。海藻酸盐作为增粘剂和凝胶成形剂具有广泛的应用前景，在许多工业领域引起了极大的兴趣。由于藻酸盐生物合成的大部分细节是已知的，以及这一途径与棕色拟青霉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高产菌株。此外，基因修饰允许影响所产生的藻酸盐的物理和化学性质。此外，以前的研究表明，在极低的溶氧压(微需氧条件)下，可以同时控制棕色拟青霉产生的海藻酸盐的分子质量和乙酰化程度。因此，这项建议的最优先事项是调查微好氧条件的潜力，以显着地最大限度地利用转基因菌株形成具有特定化学特征的藻酸盐。藻酸盐的形成导致发酵液的非牛顿流体行为，对混合的均匀性、液体中的功率输入、散热以及总体上对结果的重现性有显著影响。因此，从摇瓶到搅拌釜式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、实验室规模的搅拌釜式反应器以及50 L中试规模的加压反应器上的工艺放大和结果的再现也将是这一提议的重要部分。此外，该项目将为学生和科学家提供在墨西哥组和德国组进行研究的机会，其中墨西哥组为UNAM生物技术研究所的Peña博士，德国组为RWTH Aachen大学的Regestein博士。

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.