Glycolysis, or its variations, is a fundamental metabolic pathway in life that functions in almost all organisms to decompose external or intracellular sugars. The pathway involves the partial oxidation and splitting of sugars to pyruvate, which in turn is decarboxylated to produce acetyl-coenzyme A (CoA) for various biosynthetic purposes. The decarboxylation of pyruvate loses a carbon equivalent, and limits the theoretical carbon yield to only two moles of two-carbon (C2) metabolites per mole of hexose. This native route is a major source of carbon loss in biorefining and microbial carbon metabolism. Here we design and construct a non-oxidative, cyclic pathway that allows the production of stoichiometric amounts of C2 metabolites from hexose, pentose and triose phosphates without carbon loss. We tested this pathway, termed non-oxidative glycolysis (NOG), in vitro and in vivo in Escherichia coli. NOG enables complete carbon conservation in sugar catabolism to acetyl-CoA, and can be used in conjunction with CO2 fixation and other one-carbon (C1) assimilation pathways to achieve a 100% carbon yield to desirable fuels and chemicals.
Citreoviridin (1) belongs to a class of F1-ATPase β-subunit inhibitors that are synthesized by highly reducing polyketide synthases. These potent mycotoxins share an α-pyrone polyene structure, and they include aurovertin, verrucosidin, and asteltoxin. The identification of the citreoviridin biosynthetic gene cluster in Aspergillus terreus var. aureus and its reconstitution using heterologous expression in Aspergillus nidulans are reported. Two intermediates were isolated that allowed the proposal of the biosynthetic pathway of citreoviridin.
Fungal nonribosomal peptide synthetases (NRPSs) are megasynthetases that produce cyclic and acyclic peptides. In Aspergillus nidulans, the NRPS ivoA (AN10576) has been associated with the biosynthesis of grey-brown conidiophore pigments. Another gene, ivoB (AN0231), has been demonstrated to be an N-acetyl-6-hydroxytryptophan oxidase that putatively acts downstream of IvoA. A third gene, ivoC, has also been predicted to be involved in pigment biosynthesis based on publicly available genomic and transcriptomic information. In this paper, we report the replacement of the promoters of the ivoA, ivoB, and ivoC genes with the inducible promoter alcA in a single cotransformation. Co-overexpression of the three genes resulted in the production of a dark-brown pigment in hyphae. In addition, overexpression of each of the Ivo genes, ivoA-C, individually or in combination, allowed us to isolate intermediates and confirm the function of each gene. IvoA was found to be the first known NRPS to carry out the acetylation of the amino acid, tryptophan.
Fungal
natural products (NPs) comprise a vast number of bioactive
molecules with diverse activities, and among them are many important
drugs. However, the yields of fungal NPs from native producers are
usually low, and total synthesis of structurally complex NPs is challenging.
As such, downstream derivatization and optimization of lead fungal
NPs can be impeded by the high cost of obtaining sufficient starting
material. In recent years, reconstitution of NP biosynthetic pathways
in heterologous hosts has become an attractive alternative approach
to produce complex NPs. Here, we present an efficient, cloning-free
strategy for the cluster refactoring and total biosynthesis of fungal
NPs in Aspergillus nidulans. Our platform places
our genes of interest (GOIs) under the regulation of the robust asperfuranone afo biosynthesis gene machinery, allowing for their concerted
activation upon induction. We demonstrated the utility of our system
by creating strains that can synthesize high-value NPs, citreoviridin
(1), mutilin (2), and pleuromutilin (3), with good to high yield and purity. This platform can
be used not only for producing NPs of interests (i.e., total biosynthesis)
but also for elucidating cryptic biosynthesis pathways.
Fungal natural products comprise a wide range of bioactive
compounds
including important drugs and agrochemicals. Intriguingly, bioinformatic
analyses of fungal genomes have revealed that fungi have the potential
to produce significantly more natural products than what have been
discovered so far. It has thus become widely accepted that most biosynthesis
pathways of fungal natural products are silent or expressed at very
low levels under laboratory cultivation conditions. To tap into this
vast chemical reservoir, the reconstitution of entire biosynthetic
pathways in genetically tractable fungal hosts (total heterologous
biosynthesis) has become increasingly employed in recent years. This
review summarizes total heterologous biosynthesis of fungal natural
products accomplished before 2020 using Aspergillus nidulans as heterologous hosts. We review here Aspergillus transformation, A. nidulans hosts, shuttle vectors
for episomal expression, and chromosomal integration expression. These
tools, collectively, not only facilitate the discovery of cryptic
natural products but can also be used to generate high-yield strains
with clean metabolite backgrounds. In comparison with total synthesis,
total heterologous biosynthesis offers a simplified strategy to construct
complex molecules and holds potential for commercial application.
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