Enhancement of biofuel production in microalgae by genetic and physiological modification

Abstract

Microalgae are photosynthetic microorganisms, which could be used as potential microbial cell factories by directly converting CO2 into valuable bioproducts and biofuels. This study aims to improve the target biofuel feedstocks from the isolated green alga Chlorella sp. and the engineered cyanobacterium Synechococcus elongatus PCC 7942 in terms of the improvement on lipids (as precursors of FAMEs) and terpene (α-farnesene), respectively. The first part is concerned with the enhancement of lipids and the determination of FAMEs and biodiesel properties in Chlorella sp. under the phosphorus (P) limitation (0-50%) alone or in combination with heavy metals (Fe, Co, Pb) supplementation. The results showed that the highest yield of lipids was achieved under 0%P with 17 mM Co addition with 19% higher than the control. Moreover, the addition of low Pb concentrations could elevate the cell growth even under P limitation whereas the MUFAs, particularly palmitoleic acid (C16:1), was higher than PUFAs under most conditions. The overall biodiesel properties of the obtained FAMEs were of acceptable quality according to the standards (ASTM and EN). Additionally, the energy conversion from light energy to lipids was shown to be in the range of 10-16% conversion efficiency within 7 days, which corresponded to mixotrophic condition of microalgae cultures from previous studies. Hence, the physiological modification by stress treatments to cultures could offer the improvement of lipid content in microalgae although the genome database was not analyzed. Cyanobacterium S. elongatus PCC 7942 with available genomic database has advantages on genetic manipulation over the isolated Chlorella sp., thus the genetic modification was performed on S. elongatus PCC 7942 in this study. Herein, S. elongatus PCC 7942 was metabolically engineered for an enhanced production of α-farnesene by optimizing the ribosome-binding site (RBS) of the codon-optimized farnesene synthase gene. The production of α-farnesene was found to be enhanced in strains with a low translation initiation rate, resulting in α-farnesene productivity of 0.57 mg/L/day. Using the RBS variants and random mutation, the fluorescence-based analysis was done on cells grown in 96-well culture plates to screen the α-farnesene-producing strains, and the results showed no improvement in the titers by the RBS-optimized strains. However, evolutionary engineering of the RBS-optimized strains resulted in a two-fold increase in α-farnesene productivity (1.2 mg/L/day) compared to the previous study. Therefore, combining metabolic and evolutionary engineering might be helpful for enhancing the cellular fitness of cyanobacteria for the production of target chemicals.