Identification of Oxygen-Independent Pathways for Pyridine Nucleotide and Coenzyme A Synthesis in Anaerobic Fungi by Expression of Candidate Genes in Yeast

Abstract

NAD (NAD + ) and coenzyme A (CoA) are central metabolic cofactors whose canonical biosynthesis pathways in fungi require oxygen. Anaerobic gut fungi of the Neocallimastigomycota phylum are unique eukaryotic organisms that adapted to anoxic environments.

Pathway engineering strategies for improved product yield in yeast-based industrial ethanol production

“…Improving ethanol yield on fermentable sugars is by no means the only target of metabolic engineering studies related to yeast-based ethanol production. Other targets of intensive research include the reduction of processing costs by expression of polysaccharide hydrolases [ 136 ], extending substrate range to convert more fermentable substrates in crude industrial media [ 137 , 138 ], improving performance at high temperature to improve heat economy and cope with process temperature profiles [ 139 ], increasing yeast tolerance to process inhibitors and ethanol [ 139 , 140 ], improving osmotolerance of engineered strains with reduced glycerol formation [ 16 , 141 ] and simplification of nutritional requirements of industrial strains [ [142] , [143] , [144] , [145] ]. In addition, integration of corn-fiber from 1.5G processes [ 21 ] and reducing the need for antibiotics [ 139 , 146 ] are actively explored.…”

An engineered non-oxidative glycolytic bypass based on Calvin-cycle enzymes enables anaerobic co-fermentation of glucose and sorbitol by Saccharomyces cerevisiae

“…Genomic DNA extracted with a Qiagen Blood & Cell Culture NDA kit and 100/G Genomics-tips (Qiagen, Hilden, Germany) was quantified with a Qubit Fluorometer 2.0 (Thermo Fisher). The genome of strain IMX1489 was sequenced in-house as described previously [ 64 ] on an Illumina Miseq sequencer (Illumina, San Diego, CA, USA) with a minimum of 50-fold read coverage. Custom paired-end sequencing of genomic DNA of duplicate chemostat cultures of IMX1489 was performed by Macrogen (Amsterdam, The Netherlands) on a 350-bp PCR-free insert library using Illumina SBS technology.…”

“…Genomic DNA of strain IMX2506 was sequenced by Genomescan (Leiden, The Netherlands) with Illumina SBS technology yielding 151 bp reads with at least 50-fold read coverage. Sequence reads were mapped against the genome of S. cerevisiae CEN.PK113-7D [ 65 ] to which a virtual contig containing the sequences of pDAN-prk-tPGK1, pTDH3-cbbm-tCYC1, pTEF1-groEL-tACT1 and pTPI1-groES-tPGI1 had been added, and processed as described previously [ 64 ].…”

Anaerobic Fungal Mevalonate Pathway Genomic Biases Lead to Heterologous Toxicity Underpredicted by Codon Adaptation Indices

“…Optimization strategies with existing genes, such as fed-batch fermentations and metabolic engineering, have yielded mevalonate yields up to 30 g/L [ 56 ]. These P. indianae sourced genes may prove to confer distinct advantages through anaerobic catalytic mechanisms that improve titers further [ 7 ]. In contrast to SC.HMGR , sequence homology analysis of PI.HMGR predicts that it uses NADH rather than NADPH as a cofactor in the synthesis of mevalonate ( Figure 3 A).…”

“…Genomic studies of anaerobic fungi show they have the largest array of biomass-degrading enzymes among fungi, which can be used for platforms that convert lignocellulosic waste to biofuels and chemicals [3,5]. In addition to plant biomassdegrading enzymes, anaerobic fungi have specialized membrane transporters [6] and unique biosynthetic pathways [7,8] that may be a valuable source of natural products, like polyketides synthases (PKS), non-ribosomal peptide synthetases (NRPS), terpenes, and bacteriocins [1,9]. However, the full potential of anaerobic fungi remains unrealized because there are few existing tools for their engineering.…”