The global accumulation of petroleum-based plastics, coupled with the vast underutilization of chitin-rich shellfish waste, underscores the need for microbial platforms capable of converting renewable waste streams into biopolymers. Cupriavidus necator, a leading producer of polyhydroxyalkanoates (PHAs), cannot naturally degrade chitin, whereas Serratia marcescens possesses a highly efficient chitinolytic system that allows efficient degradation of chitin. This project aims to engineer C. necator with these S. marcescens genes to enable extracellular chitin depolymerization and subsequent assimilation of N-acetyl-D-glucosamine (GlcNAc) for PHA biosynthesis. The cloned expression vector was created and evaluated in E. coli and subsequently in C. necator to assess functional secretion and chitinase activity using colloidal chitin agar. Together, these experiments aim to permit the transformation of chitin waste into biodegradable bioplastic precursors.
- Heterologous expression of Serratia marcescens chitinolytic genes in Cupriavidus necator for chitin-derived bioplastic production
- Aiden John Morgado
- 0009-0002-4108-1209
- Christopher J Brigham (Advisor) - University of Massachusetts Dartmouth, Department of BioengineeringTracie L. Ferreira (Committee Member) - University of Massachusetts Dartmouth, Department of BioengineeringMilana C Vasudev (Committee Member) - University of Massachusetts Dartmouth, Department of Bioengineering
- ix, 54 pages]
- illustrations (chiefly color)
- List of figures -- List of tables -- Chapter 1. Background and significance -- Environmental burden of petroleum plastics and introduction to PHAs -- Cupriavidus necator as a platform for PHA production -- Chitin as an underutilized renewable feedstock -- Chitin metabolism in Serratia marcescens -- Conferring chitinolytic activity in other organisms -- Genetic and physiological considerations for heterologous expression -- Chapter 2. Problem statement and specific aims -- Problem statement -- Specific aims -- Chapter 3. Materials and methods -- Strains and plasmids -- Cloning material preparation -- Stage 1: amplification and assembly of the native chitinolytic locus -- Stage 2: Chitobiase amplification and second-stage isolation -- Preparation of electrocompetent C. Necator cells and electroporation -- Experimental media preparation -- Colloidal chitin plate assays -- Chapter 4. Results -- Design and cloning of Stage 1 construct -- Design and cloning of stage 2 construct -- C. necator transformation --Colloidal chitin growth determination -- Chapter 5. Discussion, future directions, and conclusion -- Aim 1 discussion -- Aim 2 discussion -- Aim 3 discussion -- Central limitations and observations -- Future directions -- Conclusions -- References.
- Includes bibliographical references (pages 52-54).
- University of Massachusetts Dartmouth
- Master of Science (MS)
- Biomedical Engineering and Biotechnology
- Department of Bioengineering
- English
- Thesis
- Copyright 2026 Aiden John Morgado
- https://doi.org/10.62791/20588
- 9914540173901301