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Removal of Acc1 negative regulation by mutating three phosphorylation sites;
Light-inducible overexpression of enzymes Pah1 and Dga1, which are involved in TAG synthesis;
Light-activated induction of Plin3, which supports assembly of lipid droplets;
Knockouts of lipid-degrading enzymes Tgl3, Tgl4, Tgl5;
Inactivation of the oxidative portion of pentose phosphate pathway (PPP) to allow higher carbon flux towards lipid biosynthesis;
Creating the yeast-indium phosphide nanoparticle biohybrid to use light for NADPH regeneration;
Decoupling of the growth and production phases by using light to induce a metabolic switch;
Improving yeast cell self-lysis system by anchoring glucanases to the cell wall.
A critical step in bioproduction is providing a sufficient supply of precursor molecules for the final product. The precursor for lipid synthesis is malonyl-CoA, which is synthesized from acetyl-CoA by acetyl-CoA carboxylase Acc1. It has been shown that one bottleneck in fatty acid production in S. cerevisiae is the tight posttranslational regulation of Acc1 activity by Snf1. Introduction of three mutations in Acc1 phosphorylation sites S659A, S686A, and S1157A disrupts the downregulation of Acc1 activity and has been found to lead to an increase in the malonyl-CoA abundance (Chen et al. 2018). Therefore, our team replaced wild-type ACC1 with ACC1S659A,S1157A,S686A to increase the precursor supply.
Next, we aimed to increase the production of fatty acids by overexpressing yeast genes PAH1 and DGA1, and human gene PLIN3. Pah1 and Dga1 catalyse the final steps of TAG synthesis and it has been shown that their overexpression increases TAG accumulation in S. cerevisiae (Teixeira et al. 2018). The human protein Plin3 covers the lipid droplets (LDs) and is expected to stabilize TAG by promoting LD assembly. LDs are dynamic storage organelles emerging from the endoplasmic reticulum (ER) and they stay connected to ER throughout their biogenesis and life cycle. The hydrophobic core of LD consists of TAGs that are surrounded by several layers of SEs. The core is enclosed by a phospholipid monolayer covered by perilipin proteins. LDs have contact sites with other cellular organelles, such as Golgi apparatus and mitochondria. These contacts facilitate the exchange of lipids, ions and metabolites (Athenstaedt, 2010).
To optimize the production process, we opted for inducible expression of these proteins, as lipid synthesis is energetically costly and high lipid accumulation could decrease the growth rate. We chose to use light for transcriptional activation of these genes, as light induction is robust and easily-controlled. Also, it is more eco-friendly compared to other induction systems, as it does not require addition of any compounds to the growth medium. We constructed a plasmid with three overexpression cassettes, where each gene (PAH1, DGA1, and human PLIN3) is under the control of a promoter that is regulated by VP-EL222. VP-EL222 is a blue light activated transcription factor from Erythrobacter litoralis. The promoters used in this system are inactive in dark, but are activated when the cells are exposed to blue light (Benzinger and Khammash, 2018).
Since lipids have an important structural and functional role in the cell, TAGs accumulated in LDs can be hydrolyzed and released upon requirement. S. cerevisiae uses these degradation products mainly as building blocks for membrane lipid synthesis (Czabany, Athenstaedt, and Daum, 2007). Downregulation of lipid turnover is important to achieve high lipid titers. TAG-hydrolysis is mainly driven by TAG lipases Tgl3, Tgl4 and Tgl5 (Athenstaedt and Daum, 2005). As the deletion of these genes results in increased TAG accumulation (Ploier et al., 2013; Ferreira et al., 2018), we aimed to delete TGL3, TGL4 and TGL5 in our production strain.
In addition to malonyl-CoA, each step of fatty acid elongation by two carbon units requires 2 NADPH molecules. NADPH is produced in the cytosol via pentose phosphate pathway (PPP). The initial step of PPP is catalyzed by glucose-6-phosphate dehydrogenase (Zwf1 in S. cerevisiae) (He et al., 2018; Sheng and Feng, 2015). However, when a hexose sugar is oxidized in PPP, two equivalents of CO2 are lost, which leads to a decrease in theoretical carbon yield. In our project, we aimed to decouple NADPH generation from the central carbon metabolism and thus maximize the carbon flux towards lipid biosynthesis. We have knocked out the ZWF1 gene to disrupt the oxidative portion of PPP and redirect more carbon towards synthesis of the final product. However, simple inactivation of PPP would inevitably lead to decreased availability of cytosolic NADPH. An alternative supply for the cytosolic NADPH has been suggested by Guo et al. (2018). The scientists have created a semiconductor biohybrid system by functionalizing genetically engineered yeast cells with light-absorbing indium phosphide (InP) nanoparticles. There are two reasons for using InP as the photosensitizer. First, the optical and electronic properties of nanoscale InP enable efficient absorption of a large fraction of the solar spectrum (Guo et al., 2018). Secondly, InP is by far one of the most biocompatible and sustainable semiconductors in biohybrid engineering. Taken together, these two factors make it an ideal material for solar-driven biocatalysis. Utilizing light for NADPH regeneration increases the lipid production efficiency, as more carbon can be directed to the final product.
To increase the efficiency of the production process, we decided to unlink growth and lipid accumulation phases. It has been shown that simple overproduction of lipid-synthesizing enzymes impedes cell growth. More specifically, endogenous pathways that are essential for cell growth may compete with lipid biosynthesis, especially following the exhaustion of carbon source in the culture medium (Papanikolaou and Aggelis, 2011; Zhao et al., 2018). This competition, in turn, leads to decreased biomass and reduced production efficiency. To address this, scientists frequently use inducible systems to uncouple the competing pathways (Zhao et al., 2018). This approach enables to separate a growth phase, when additional lipid biosynthesis genes are not expressed, and a production phase, when carbon flux to the lipid biosynthesis pathway is maximized. Our designed bioreactor allowed us to grow yeast-InP biohybrids first in the presence of red light, which activates the InP nanoparticles but does not induce the overexpression cassettes, and after reaching certain cell density, the lipid biosynthesis can be activated with blue light.
Downstream processing is an integral part of the production process and it often laborious and costly. It has been estimated that product extraction and purification often takes about 85% of total costs in a biomanufacturing process. In addition to being energy-intensive, lipid extraction involves the use of high amounts of toxic solvents, as it requires the disruption of the cell wall (Yu et al. 2015). When the lipids are manufactured for food industry, toxic chemicals should be avoided. Taken together, the cost and the complexity of downstream processing are two critical factors that make bioproduction less competitive in comparison to chemical synthesis (Renneberg, Berkling and Loroch, 2016).
Lipid extraction from wet biomass would significantly reduce the energy spent on dewatering the cell biomass. However, the available technologies are far from being commercialized (Dong et al. 2016). An easier and less expensive process of lipid recovery, for example, by excretion of lipids to the medium, would make lipid bioproduction more competitive (Vasconcelos et al. 2019). Our team has previously shown that induced expression of bacterial glucanases has a potential to cause cell lysis. In that project, the glucanases were secreted from the cells. However, the efficiency of autolysis could be improved by increasing the local concentration of glucanases in the cell wall. The bacterial glucanases could potentially be anchored to the yeast cell wall by fusion with Glycosylphosphatidylinositol(GPI)-anchored cell wall proteins (Inokuma et al., 2020). To ease lipid extraction from yeast cells, we aimed to improve the inducible yeast autolysis by overexpressing β-1,3-glucan laminaripentao-hydrolase (Glc1) and endo-1,3-β-glucanase from Cellulosimicrobium cellulans (BBa_K2711000) fused to GPI-anchors.
We planned to test the lipid production capability of all constructed yeast strains by measuring the lipid content at different Cultivation time points using Nile Red staining. Nile Red is a fluorescent stain that specifically binds to the neutral lipids and can be used to measure lipid droplets content (Greenspan, Mayer, and Fowler, 1985). The Nile Red staining was measured both at a culture level using a microplate reader and also at a single cell level using fluorescence microscopy.
In addtition, the inducible cell lysis will be monitored in time-lapse microscopy experiments, where cells are grown to sufficient density, followed by light-dependent induction of the glucanase genes. This approach allows both detection of cell lysis and any other morphological changes caused to the cell by induction of the glucanase genes. In the final production strain, when we combine autolysis and lipid production, the inductor for autilysis will be changed.
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