Name: Abdulmajid Alrefaie
Date: 08/28/2026
Time (EST/EDT): 10:00 am
Location: Institute of Marine and Environmental Technology (IMET) - Multipurpose Room (MPR)
Remote Access: email: mees@umd.edu

Committee Chair: Dr. Yantao Li
Committee Members: Dr. Allen Place, Dr. Tsvetan Bachvaroff, Dr. Stephen Miller, and Dr. Guanqun Chen
Dean’s Representative: Dr. Birthe Veno Kjellerup Shirtliff

Title: Understanding Heterotrophic Acetate Metabolism in Chlamydomonas reinhardtii

Abstract: The unicellular green alga Chlamydomonas reinhardtii (C. reinhardtii) uses acetate as its sole organic carbon source during heterotrophic growth; however, the functions of genes involved in acetate catabolic pathways remain unclear. This dissertation investigated insertional mutants of acetyl-CoA synthetase 1 (ACS1) and acetate kinase 2 (ACK2) using physiological, biochemical, transcriptomic, and genetic approaches. Under heterotrophic conditions, the acs1 mutant reached a 29.8% higher cell density than the wild-type strain CC-5325 at 96 h and exhibited a 38.3% lower triacylglycerol (TAG) content at 48 h. In contrast, ack2 reached a 27.7% lower cell density and exhibited delayed oxygen consumption, whereas its biomass concentration and dry weight per cell were 39.3% and 90.4% higher, respectively, at 96 h. At 120 h, ack2 produced 23.3% more total carbohydrate and approximately 2.2-fold more starch per cell. RNA sequencing revealed distinct transcriptomic responses under heterotrophic conditions. At 24 h, acs1 exhibited 2,226 differentially expressed genes (DEGs), compared with 4,615 in ack2. In ack2, the glycolysis-associated genes glucokinase 1 (GLK1), phosphofructokinase 1 (PFK1), glyceraldehyde-3-phosphate dehydrogenase 2 (GAP2), and pyruvate kinase 2 (PYK2) were downregulated by approximately 2.8-, 3.2-, 9.2-, and 4.3-fold, respectively, relative to CC-5325. Several tricarboxylic acid (TCA) cycle genes were also downregulated. Within starch metabolism, the starch biosynthesis and remodeling genes ADP-glucose pyrophosphorylase 2 (AGP2) and isoamylase 1 (ISA1) were upregulated 3.73- and 3.25-fold, respectively, whereas the starch degradation gene β-amylase 2 (AMB2) was downregulated by 11.3-fold. At 48 h, acs1 exhibited an altered TAG fatty acid (FA) composition, with saturated FAs accounting for 70.2% of total TAG FAs, compared with 41.8% in CC-5325. To examine the combined genetic effects of ACS1 and ACK2 mutant, progeny of opposite mating types carrying individual mutations were crossed and screened for acs1 ack2 double mutants. Despite extensive crossing and screening, no purified double mutant was confirmed. Together, these findings indicate that disruption of ACS1 and ACK2 contributes differently to acetate-dependent carbon partitioning in C. reinhardtii. Loss of cytosolic ACS1 is associated with increased cell proliferation and reduced TAG accumulation, suggesting a shift away from lipid biosynthesis toward growth. In contrast, loss of mitochondrial ACK2 suppresses glycolytic and TCA-cycle gene expression while promoting starch accumulation and increasing biomass per cell, consistent with a redirection of carbon flux from respiratory metabolism toward carbohydrate storage. These results support compartment-specific roles for acetate catabolism in coordinating growth and carbon allocation during heterotrophic metabolism.