Designer cell refactoring and organelle remodeling for highly enhanced terpene production

Abstract

As an engineering discipline for biological systems, the synthetic biology aims to design microbial cell factories towards efficient production of high-value products. The redesigned microorganisms permit a directed modification of metabolic pathways to rewire cellular metabolism for the synthesis of pharmaceuticals, chemicals, fuels, and other valuable products. However, the engineered metabolic pathways present several challenges, including unfavorable regulatory responses, suboptimal physicochemical environments, loss of intermediates to competing pathways, and metabolite toxicity. In achieving optimal metabolic reactions or supporting unique physiological functions, harnessing subcellular organelles would be a promising strategy to address current limitations and future possibilities. Indeed, many researchers have devised the strategies that enhance the limited synthetic capacity of cells by remodeling cell functions to manipulate an intrinsic organelle-specific activity, shape, and capacity (e.g., size, number, and volume) and introducing new functionality to the cells for the production of natural and even non-natural products. Here, we present two research projects for employing synthetic biology to design and refactor the cell with new functionality for the enhanced synthesis of high-value products. In the first study, we redesign lipid droplets (LDs) which is not a simple blob of fat but a highly dynamic organelle capable of regulating lipid metabolism, storage, and transportation. In guiding LDs to serve as storage vessels that insulate high-value lipophilic compounds in cells, we demonstrate that chain flexibility of lipids determines their selective migration in intracellular LDs. Focusing on commercially important medicinal lipids (e.g., terpene such as squalene and zeaxanthin) with biogenetic similarity but structural dissimilarity, we experimentally validate that LD remodeling should be differentiated between overproduction of structurally flexible squalene and that of rigid zeaxanthin and β-carotene. Among medicinal terpenes, flexible squalene showed dramatically increased intracellular storage, by ~3100%, due to LD enlargement, while rigid zeaxanthin and β-carotene benefited from LD. In conclusion, we verify that intracellular storage of structurally flexible squalene significantly increases with LD volume expansion, but that of rigid zeaxanthin and β-carotene is enhanced through LD surface broadening. Importantly, this LD engineering strategy can be applied to the storage of non-saponifiable lipids and to other types of lipid transport systems and even metabolic systems. In the second research project, by utilizing well-characterized signal tags which can accurately deliver proteins to intracellular organelles or extracellular milieu, we construct synthetic pathway for a metabolite trafficking system with a novel functionality that enables hydrophobic impermeable-product secretion by yeast cells. For this study, we systematically couple a supernatant binding protein, which is cytosolic lipid-binding protein as a large and hydrophobic medicinal terpene carrier, with an export signal peptide. By the novel metabolite trafficking system, terpenes are successfully secreted out of the cells (~225 mg/L for squalene and ~1.6 mg/L for β-carotene). This sustainable metabolite secretion holds a great potential for mass production of high-value chemicals in an efficient and continuous manner by continuedly extracting the bio-based chemicals from extracellular medium without cell disruption. Thus, we implemented a semi-continuous fermentation to prove that our metabolite trafficking system could be used to produce and secrete the target metabolite in a continuous and efficient manner (~670 mg/L for squalene for 15-day semi-continuous culture). To the best of our knowledge, this is the most efficient cognate pathway for selective metabolite secretion in microorganisms, thus enabling the intracellularly-accumulated target compounds to pass through otherwise impermeable membranes.