Bioengineering a non-pathogenic bacteria to produce medically-relevant biopolymers

 描述（由申请人提供）：藻酸盐是一种商业上可行的阴离子生物聚合物，由两种单体亚基α-D-甘露糖醛酸盐和α-L-古洛糖醛酸盐组成。藻酸盐用于各种应用，包括纺织品制造、废水治理食品生产以及各种医疗应用，如伤口护理和药物输送。目前，商业藻酸盐是从各种属的褐色海藻中收获的。然而，海藻衍生的藻酸盐具有若干限制，包括：季节性生长，收获之间产量、组成和质量的不一致，供应方不稳定性以及使用经济和环境昂贵的加工方法。这些限制影响海藻衍生的藻酸盐用于生物医学和制药应用。藻酸盐也由固氮菌属和假单胞菌属的细菌产生。此外，细菌衍生的藻酸盐具有几个优点;具体地说，它相对容易，便宜，并且对培养和收获环境友好。此外，细菌可以很容易地操作，以产生大量的海藻酸盐，具有特定的定制特性，用于生物医学和制药应用。然而，很少有正在进行的研究来开发用于商业化藻酸盐生产的细菌菌株。Progenesis Technologies，LLC已获得用于重组藻酸盐生产的稳定的高藻酸盐生产铜绿假单胞菌菌株的专利。在一次请愿前咨询中，美国联邦药品管理局（FDA）建议废除分泌的毒力因子外毒素A和绿脓菌素的功能，以降低VE 2的相对致病性。FDA还建议使用目前批准的菌株生产藻酸盐。到目前为止，我们已经废除了外毒素A，绿脓菌素，磷脂酶C和内毒素的毒力因子的功能。在这个提议中，我们打算设计和评估一种用于生产生物聚合物藻酸盐的非致病性细菌菌株。我们建议通过完成两个具体目标来实现这一目标。在目的1中，我们将使用鼠模型评估我们最近修饰的铜绿假单胞菌菌株PGN 4的致病性。在目标2中，我们将确定使用目前批准的E.大肠杆菌菌株。在项目结束时，我们将寻求适当的联邦批准，使用我们新获得的非致病性菌株作为生产生物医学和药用级海藻酸盐的平台，用于各种医疗应用。

 DESCRIPTION (provided by applicant): Alginate is a commercially-viable anionic biopolymer consisting of two monomeric subunits, ß-D-mannuronate and a-L-guluronate. Alginate is used in a variety of applications including textile manufacturing, waste water remediation food production, and a variety of medical applications such as wound care and drug delivery. Currently, commercial alginate is harvested from various genera of brown seaweeds. However, seaweed- derived alginate has several limitations including: seasonal growth, inconsistencies in yield, composition and quality between harvests, supply-side instability and the use of economically and environmentally costly processing methods. These limitations impact seaweed-derived alginate's use for biomedical and pharmaceutical application. Alginate is also produced by bacteria in the genera Azotobacter and Pseudomonas. Moreover, bacteria-derived alginate has several advantages; specifically, it is relatively easy, inexpensive, and environmentally-friendly to cultivate and harvest. Additionally, the bacteria can be easily manipulated to produce large amounts of alginate with specific customized properties for biomedical and pharmaceutical applications. However, there is little ongoing research to develop bacterial strains for commercial alginate production. Progenesis Technologies, LLC has patented a stable high alginate producing strain of Pseudomonas aeruginosa for use in recombinant alginate production. At a pre-petition consultation, the United States Federal Drug Administration (FDA) suggested abrogating the function of the secreted virulence factors exotoxin A and pyocyanin to decrease the relative pathogenicity of VE2. The FDA also suggested using the currently approved bacterial strains for alginate production. To date, we have abrogated the function of the virulence factors exotoxin A, pyocyanin, phospholipase C and endotoxin. In this proposal, we intended to design and evaluate a non- pathogenic bacterial strain for the production of the biopolymer alginate. We propose to accomplish this through the completion of two Specific Aims. In Aim 1, we will evaluate the pathogenicity of our recently modified P. aeruginosa strain PGN4 using a murine model. In Aim 2, we will determine the feasibility of producing alginate using currently approved E. coli strains. At the conclusion of ths project, we will seek the appropriate federal approval to use our newly-acquired, non-pathogenic bacterial strain as a platform for the production of biomedical and pharmaceutical grade alginate for use in various medical applications.