The eccDNA replicon enables glyphosate resistance in Amaranthus palmeri One-sentence summary: The eccDNA replicon is a large extra-nuclear circular DNA that is composed of a sophisticated repetitive structure, harbors the EPSPS and several other genes that are transcribed during glyphosate stress.

BackgroundThe oleaginous yeast, Yarrowia lipolytica, has been utilized as an industrial host for about 60 years for various applications. Recently, the metabolic engineering of this host has become increasingly popular due to its ability to accumulate lipids as well as improvements made toward developing new genetic tools. Y. lipolytica can robustly metabolize glucose, glycerol, and even different lipid classes. However, little is known about its xylose metabolizing capability. Given the desirability of having a robust xylose utilizing strain of Y. lipolytica, we performed a comprehensive investigation and elucidation of the existing components of its xylose metabolic pathway.ResultsA quick and efficient means of determining functionality of the candidate xylose pathway genes (XYR, XDH, and XKS) from Y. lipolytica was desirable. We challenged Escherichia coli mutants lacking either the xylose isomerase (xylA) gene or the xylulose kinase (xylB) gene to grow on xylose minimal media by expressing the candidate genes from Y. lipolytica. We showed that the XKS of Y. lipolytica is able to rescue xylose growth of E. coli ΔxylB, and the XDH enabled growth on xylitol, but not on xylose, of E. coli ΔxylA. Overexpression of XKS and XDH in Y. lipolytica improved growth on xylitol, indicating that expression of the native enzymes was limiting. Overexpression of XKS and XDH in Y. lipolytica also enables robust growth on xylose under high nitrogen conditions without the need for adaptation. These results prove that a complete xylose pathway exists in Y. lipolytica, but the pathway is poorly expressed. To elucidate the XYR gene, we applied the E. coli ΔxylA xylose growth challenge with 14 candidate XYR genes and XDH. The XYR2 candidate was able to rescue growth of E. coli ΔxylA xylose on minimal media.ConclusionsWhile a native xylose pathway exists in Y. lipolytica, the microorganism’s inability to grow robustly on xylose is an effect of cryptic genetic circuits that control expression of key enzymes in the metabolic pathway. We have characterized the key enzymes associated with xylose metabolism and demonstrated that gene regulatory issues can be overcome using strong hybrid promoters to attain robust growth on xylose without adaptation.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0562-6) contains supplementary material, which is available to authorized users.

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There is growing interest in using oleaginous yeast for the production of a variety of fatty acids and fatty acid-derived oleochemicals. This is motivated by natural propensity for high flux through lipid biosynthesis that has naturally evolved, making them a logical starting point for additional genetic engineering to improve titers and productivities. Much of the academic and industrial focus has centered on yeast that have significant genetic engineering tool capabilities, such as Yarrowia lipolytica , and those that have naturally high lipid accumulation, such as Rhodosporidium toruloides and Lipomyces starkeyi ; however, there are oleaginous yeast with phenotypes better aligned with typically inhibitory process conditions, such as high salt concentrations and lignocellulosic derived inhibitors. This review addresses the foundational work in characterizing two emerging oleaginous yeast of interest: Debaryomyces hansenii and Trichosporon oleaginosus . We focus on the physiological and metabolic properties of these yeast that make each attractive for bioprocessing of lignocellulose to fuels and chemicals, discuss their respective genetic engineering tools and highlight the critical barriers facing the broader implementation of these oleaginous yeast.

BackgroundThe oleaginous yeast, Trichosporon oleaginosus, has been extensively studied for its ability to metabolize non-conventional feedstocks. These include phenol-containing waste streams, such as distillery wastewater, or streams consisting of non-conventional sugars, such as hydrolyzed biomass and various bagasse. An initial BLAST search suggests this yeast has putative aromatic metabolizing genes. Given the desirability to valorize underutilized feedstocks such as lignin, we investigated the ability of T. oleaginosus to tolerate and metabolize lignin-derived aromatic compounds.Results Trichosporon oleaginosus can tolerate and metabolize model lignin monoaromatics and associated intermediates within funneling pathways. Growth rates and biomass yield were similar to glucose when grown in 4-hydroxybenzoic acid (pHBA) and resorcinol, but had an increased lag phase when grown in phenol. Oleaginous behavior was observed using resorcinol as a sole carbon source. Fed-batch feeding resulted in lipid accumulation of 69.5% on a dry weight basis.ConclusionsThough the exact pathway of aromatic metabolism remains to be determined for T. oleaginosus, the results presented in this work motivate use of this organism for lignin valorization and phenolic wastewater bioremediation. Trichosporon oleaginosus is the first yeast shown to be oleaginous while growing on aromatic substrates, and shows great promise as a model industrial microbe for biochemical and biofuel production from depolymerized lignin.Electronic supplementary materialThe online version of this article (10.1186/s12934-017-0820-8) contains supplementary material, which is available to authorized users.

Short Title: The eccDNA replicon enables glyphosate resistance in Amaranthus palmeri 15 16One-sentence summary: The eccDNA replicon is a large extra-nuclear circular DNA that is 17 composed of a sophisticated repetitive structure, harbors the EPSPS and several other genes that 18are transcribed during glyphosate stress.

Objective: The objective of the research presented here was to determine whether autonomous replication sequences (ARS) discovered in the eccDNA replicon of glyphosate resistant Amaranthus palmeri enable self-replication in a yeast system. Results: Sequence analysis of the eccDNA replicon revealed a region of sharp changes in A + T/G + C content with characteristic bending indicative of an autonomous replication sequence. Further sequence analysis revealed an extended autonomous replication sequence (EACS) in close proximity to multiple DNA unwinding element (DUE) sequences. This region of the eccDNA replicon enabled autonomous replication of an ARS-less yeast plasmid.

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