Single-cell genetic models to evaluate orphan gene function: The case of QQS regulating carbon and nitrogen allocation.

We demonstrate two synthetic single-cell systems that can be used to better understand how the acquisition of an orphan gene can affect complex phenotypes. The Arabidopsis orphan gene, Qua-Quine Starch (QQS) has been identified as a regulator of carbon (C) and nitrogen (N) partitioning across multiple plant species. QQS modulates this important biotechnological trait by replacing NF-YB (Nuclear Factor Y, subunit B) in its interaction with NF-YC. In this study, we expand on these prior findings by developing Chlamydomonas reinhardtii and Saccharomyces cerevisiae strains, to refactor the functional interactions between QQS and NF-Y subunits to affect modulations in C and N allocation. Expression of QQS in C. reinhardtii modulates C (i.e., starch) and N (i.e., protein) allocation by affecting interactions between NF-YC and NF-YB subunits. Studies in S. cerevisiae revealed similar functional interactions between QQS and the NF-YC homolog (HAP5), modulating C (i.e., glycogen) and N (i.e., protein) allocation. However, in S. cerevisiae both the NF-YA (HAP2) and NF-YB (HAP3) homologs appear to have redundant functions to enable QQS and HAP5 to affect C and N allocation. The genetically tractable systems that developed herein exhibit the plasticity to modulate highly complex phenotypes.

Modifying the yeast very long chain fatty acid biosynthetic machinery by the expression of plant 3-ketoacyl CoA synthase isozymes.

Eukaryotes express a multi-component fatty acid elongase to produce very long chain fatty acids (VLCFAs), which are building blocks of diverse lipids. Elongation is achieved by cyclical iteration of four reactions, the first of which generates a new carbon-carbon bond, elongating the acyl-chain. This reaction is catalyzed by either ELONGATION DEFECTIVE LIKE (ELO) or 3-ketoacyl-CoA synthase (KCS) enzymes. Whereas plants express both ELO and KCS enzymes, other eukaryotes express only ELOs. We explored the Zea mays KCS enzymatic redundancies by expressing each of the 26 isozymes in yeast strains that lacked endogenous ELO isozymes. Expression of the 26 maize KCS isozymes in wild-type, scelo2 or scelo3 single mutants did not affect VLCFA profiles. However, a complementation screen of each of the 26 KCS isozymes revealed five that were capable of complementing the synthetically lethal scelo2; scelo3 double mutant. These rescued strains express novel VLCFA profiles reflecting the different catalytic capabilities of the KCS isozymes. These novel strains offer a platform to explore the relationship between VLCFA profiles and cellular physiology.

A single-cell platform for reconstituting and characterizing fatty acid elongase component enzymes.

Fatty acids of more than 18-carbons, generally known as very long chain fatty acids (VLCFAs) are essential for eukaryotic cell viability, and uniquely in terrestrial plants they are the precursors of the cuticular lipids that form the organism's outer barrier to the environment. VLCFAs are synthesized by fatty acid elongase (FAE), which is an integral membrane enzyme system with multiple components. The genetic complexity of the FAE system, and its membrane association has hampered the biochemical characterization of FAE. In this study we computationally identified Zea mays genetic sequences that encode the enzymatic components of FAE and developed a heterologous expression system to evaluate their functionality. The ability of the maize components to genetically complement Saccharomyces cerevisiae lethal mutants confirmed the functionality of ZmKCS4, ZmELO1, ZmKCR1, ZmKCR2, ZmHCD and ZmECR, and the VLCFA profiles of the resulting strains were used to infer the ability of each enzyme component to determine the product profile of FAE. These characterizations indicate that the product profile of the FAE system is an attribute shared among the KCS, ELO, and KCR components of FAE.