Systems biology-guided understanding of white-rot fungi for biotechnological applications: A review.

Plant-derived biomass is the most abundant biogenic carbon source on Earth. Despite this, only a small clade of organisms known as white-rot fungi (WRF) can efficiently break down both the polysaccharide and lignin components of plant cell walls. This unique ability imparts a key role for WRF in global carbon cycling and highlights their potential utilization in diverse biotechnological applications. To date, research on WRF has primarily focused on their extracellular 'digestive enzymes' whereas knowledge of their intracellular metabolism remains underexplored. Systems biology is a powerful approach to elucidate biological processes in numerous organisms, including WRF. Thus, here we review systems biology methods applied to WRF to date, highlight observations related to their intracellular metabolism, and conduct comparative extracellular proteomic analyses to establish further correlations between WRF species, enzymes, and cultivation conditions. Lastly, we discuss biotechnological opportunities of WRF as well as challenges and future research directions.

Synthetic Biology towards Engineering Microbial Lignin Biotransformation.

Lignin is the second most abundant biopolymer on earth and is a major source of aromatic compounds; however, it is vastly underutilized owing to its heterogeneous and recalcitrant nature. Microorganisms have evolved efficient mechanisms that overcome these challenges to depolymerize lignin and funnel complex mixtures of lignin-derived monomers to central metabolites. This review summarizes recent synthetic biology efforts to enhance lignin depolymerization and aromatic catabolism in bacterial and fungal hosts for the production of both natural and novel bioproducts. We also highlight difficulties in engineering complex phenotypes and discuss the outlook for the future of lignin biological valorization.

Autonomous replication sequences from the Amaranthus palmeri eccDNA replicon enable replication in yeast.

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.

The EccDNA Replicon: A Heritable, Extranuclear Vehicle That Enables Gene Amplification and Glyphosate Resistance in Amaranthus palmeri.

Gene copy number variation is a predominant mechanism used by organisms to respond to selective pressures from the environment. This often results in unbalanced structural variations that perpetuate as adaptations to sustain life. However, the underlying mechanisms that give rise to gene proliferation are poorly understood. Here, we show a unique result of genomic plasticity in Amaranthus palmeri: a massive, ∼400-kb extrachromosomal circular DNA (eccDNA) that harbors the 5-ENOYLPYRUVYLSHIKIMATE-3-PHOSPHATE SYNTHASE (EPSPS) gene and 58 other genes whose encoded functions traverse detoxification, replication, recombination, transposition, tethering, and transport. Gene expression analysis under glyphosate stress showed transcription of 41 of these 59 genes, with high expression of EPSPS, as well as genes coding for aminotransferases, zinc finger proteins, and several uncharacterized proteins. The genomic architecture of the eccDNA replicon is composed of a complex arrangement of repeat sequences and mobile genetic elements interspersed among arrays of clustered palindromes that may be crucial for stability, DNA duplication and tethering, and/or a means of nuclear integration of the adjacent and intervening sequences. Comparative analysis of orthologous genes in grain amaranth (Amaranthus hypochondriacus) and waterhemp (Amaranthus tuberculatus) suggests that higher order chromatin interactions contribute to the genomic origins of the A. palmeri eccDNA replicon structure.

Identification of oleaginous yeasts that metabolize aromatic compounds.

The valorization of lignin is critical for the economic viability of the bioeconomy. Microbial metabolism is advantageous for handling the myriad of aromatic compounds resulting from lignin chemical or enzymatic depolymerization. Coupling aromatic metabolism to fatty acid biosynthesis makes possible the production of biofuels, oleochemicals, and other fine/bulk chemicals derived from lignin. Our previous work identified Cutaneotrichosporon oleaginosus as a yeast that could accumulate nearly 70% of its dry cell weight as lipids using aromatics as a sole carbon source. Expanding on this, other oleaginous yeast species were investigated for the metabolism of lignin-relevant monoaromatics. Thirty-six oleaginous yeast species from the Phaff yeast collection were screened for growth on several aromatic compounds representing S-, G-, and H- type lignin. The analysis reported in this study suggests that aromatic metabolism is largely segregated to the Cutaenotrichosporon, Trichosporon, and Rhodotorula clades. Each species tested within each clade has different properties with respect to the aromatics metabolized and the concentrations of aromatics tolerated. The combined analysis suggests that Cutaneotrichosporon yeast are the best suited to broad spectrum aromatic metabolism and support its development as a model system for aromatic metabolism in yeast.

Oleaginous yeast for biofuel and oleochemical production.

Current transportation fuels derived from petroleum can also be made from microbial systems. In particular, oleaginous yeast have naturally evolved high flux pathways for fatty acids in the form of neutral lipids, which can be converted into a variety of drop-in fuels. Here, we describe the recent advances in the use of the four most popular oleaginous yeasts for making lipids and other potential fuels - Yarrowia lipolytica, Lipomyces starkeyi, Rhodosporidium toruloides, and Cutaneotrichosporon oleaginosus. The paper is divided into three major sections focusing on (1) the important natural complex phenotypes of each yeast; (2) the development of metabolic engineering tools for each yeast; and (3) demonstrations of metabolic engineering in each yeast. At the end of each section, we provide our assessment, of which yeast is most promising in the near and long term for bioenergy production.

Engineering yeast for utilization of alternative feedstocks.

Realizing the economic benefits of alternative substrates for commodity chemical bioproduction typically requires significant metabolic engineering of common model organisms, such as Saccharomyces cerevisiae. A growing toolkit is enabling engineering of non-conventional yeast that have robust native metabolism for xylose, acetate, aromatics, and waste lipids. Scheffersomyces stipitis was engineered to produce itaconic acid from xylose. Yarrowia lipolytica produced lipids from dilute acetate at over 100g/L. Cutaneotrichosporon oleaginosus was engineered to produce omega-3 fatty acids and recently was shown to accumulate nearly 70% lipids when grown on aromatics as a carbon source. Further improvement to toolkits for genetic engineering of non-conventional yeast will enable future development of alternative substrate conversion to biochemicals.

Metabolism of aromatics by Trichosporon oleaginosus while remaining oleaginous.

BACKGROUND: The 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. CONCLUSIONS: Though 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.

New kids on the block: emerging oleaginous yeast of biotechnological importance.

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.

Engineering xylose utilization in Yarrowia lipolytica by understanding its cryptic xylose pathway.

BACKGROUND: The 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. RESULTS: A 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. CONCLUSIONS: While 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.