Source: Dissertation Abstracts International, Volume: 78-07(E), Section: B.

Adviser: Scott A. Strobel.

Humans have harnessed microorganisms and their natural products for thousands of years in a wide range of industrial, agricultural, and medical applications. However, we have only accessed a small fraction of nature's potential as a vast majority of the microorganisms on Earth have yet to be discovered. There is an increasing need to develop new, sustainable biotechnologies to keep pace with our rapidly increasing population and industrial manufacturing. The development of sustainable biotechnology relies heavily on the search for novel organisms, novel natural products, and novel enzymatic activity that can be used in practical applications.

An untapped reservoir of microbial diversity exists within vascular plants. Endophytes, microorganisms that live within the inner tissues of plants without causing overt negative symptoms, are excellent candidates for the discovery of novel biological activity because they inhabit millions of unique biological niches inside plants that are living in many different environments. Numerous novel natural products have been isolated from endophytes including soluble and volatile antibiotics, antiviral compounds, anticancer agents, antioxidants, antidiabetic agents, and immunosuppressive compounds. In this study I investigate the use of endophytic fungi for two applications in sustainable biotechnology: (i) mycofumigation and (ii) bioremediation of plastic and rubber polymers.

Modern agriculture is heavily dependent on synthetic fumigants to fight pathogenic infection of crops and produce. However, given concerns about the effects of synthetic fumigants on human health and the environment, alternative fumigation techniques are being investigated. Mycofumigation is a relatively recent biocontrol strategy that utilizes fungal volatile organic compounds to inhibit pathogenic bacteria and fungi. The term mycofumigation was coined after discovery of the endophytic fungus Muscodor albus, which exhibits potent, broad-spectrum inhibition of numerous human and plant pathogens. However, the chemical(s) responsible for toxicity and the Muscodor mechanism of action had not been elucidated.

Here I identify the mechanism of action of M. albus. By analyzing a collection of Muscodor isolates with varying toxicity, I demonstrate that the volatile compound N-methyl- N-nitrosoisobutyramide is the dominant factor in Muscodor toxicity and acts primarily through DNA methylation. Muscodor isolates also exhibit higher resistance to DNA methylation compared to other fungi. This work provides insight into chemical strategies that organisms use to manipulate their environment and provokes questions regarding the mechanisms of resistance used to tolerate constitutive, long-term exposure to DNA methylation.

Additionally, with the increasing prevalence of plastic and rubber materials in our everyday lives, it is imperative to develop better methods for their safe and efficient disposal. We have seen the emergence of a throwaway economy, where materials are constantly purchased, thrown away, and forgotten. It is no surprise that plastic and rubber products, which are designed to be resilient, accumulate in our landfills and natural environments at an alarming rate. Bioremediation is an attractive technique that utilizes microorganisms to degrade unwanted contaminants, and increasing numbers of microorganisms that target plastic and rubber degradation have been identified. Endophytic fungi are promising candidates for the degradation of plastic and rubber materials since they have uniquely-adapted enzymes to degrade natural plant polymers. To contribute to the development of cost-effective methods for the bioremediation of plastic waste it is essential to identify and characterize plastic- and rubber-degrading organisms and enzymes that could be used for industrial applications.

In this study, I characterized enzymatic degradation of the plastic polyester polyurethane (PUR) by the endophytic fungus Pestalotiopsis microspora E2712A. I recombinantly expressed the PUR-degrading enzyme, identified as a cutinase, from P. microspora and determined that this enzyme did not display outstanding enzymatic characteristics, in terms of thermal stability or kinetic esterase activity, compared cutinases from Aspergillus oryzae and Humicola insolens. I determined that P. microspora was unable to degrade PUR in anaerobic environments as previously reported. Additionally, hundreds of endophytic fungi were screened for the ability to degrade natural rubber and the more recalcitrant synthetic rubber, polychloroprene, commonly known as Neoprene. The screens included endophytes collected from the rainforests in Ecuador, endophytes collected at a local landfill, and endophytes specifically isolated from latex-producing plants. Unfortunately, no reliable rubber-degrading hits were obtained from these screens. Nevertheless, this work contributes to the understanding of plastic biodegradation by P. microspora and provides insight into the methods used to screen for fungal degradation of natural and synthetic rubber.