Metabolic engineering in woody plants: challenges, advances, and opportunities

Woody plant species represent an invaluable reserve of biochemical diversity to which metabolic engineering can be applied to satisfy the need for commodity and specialty chemicals, pharmaceuticals, and renewable energy. Woody plants are particularly promising for this application due to their low input needs, high biomass, and immeasurable ecosystem services. However, existing challenges have hindered their widespread adoption in metabolic engineering efforts, such as long generation times, large and highly heterozygous genomes, and difficulties in transformation and regeneration. Recent advances in omics approaches, systems biology modeling, and plant transformation and regeneration methods provide effective approaches in overcoming these outstanding challenges. Promises brought by developments in this space are steadily opening the door to widespread metabolic engineering of woody plants to meet the global need for a wide range of sustainably sourced chemicals and materials.

Genome Mining of Secondary Metabolites From a Marine-derived Aspergillus Terreus B12

As an important saprophytic filamentous fungus, Aspergillus terreus is ubiquitously distributed, including soil rhizospheres and marine environments. Due to the prominent capabilities of bioconversion and biosynthesis, A. terreus has become attractive in biotechnical and pharmaceutical industry. In this work, an A. terreus strain, B12, was isolated from sponge in South China Sea, which demonstrated broad bacteriostatic effects against a variety of pathogenic bacteria. The whole genome was sequenced, showing a genetic richness of BGCs, which might underpin the metabolic plasticity and adaptive resilience for the strain. Genome mining identified 67 biosynthetic gene clusters (BGCs), among which, 6 gene clusters could allocate to known BGCs (100% identity), corresponding to diverse metabolites like clavaric acid, dihydroisoflavipucine /isoflavipucine, dimethylcoprogen, alternariol, aspterric acid and pyranonigrin E. However, instead of the putative compounds, several other products were obtained from the B12 fermentation, including terrein, butyrolactone I, terretonin A&E, acoapetaline B and epi-aszonalenins A. Of note, acoapetaline B and epi-aszonalenins A, discovered natural products recently with little information, unexpectedly were reported in this A. terreus strain. The genomic and heterogeneity observed in strain B12, should be at least partially attributed to the genetic variability and biochemical diversity of A. terreus , which could be an interesting issue open to future efforts.

Biochemical unity revisited: microbial central carbon metabolism holds new discoveries, multi-tasking pathways, and redundancies with a reason

For a long time, our understanding of metabolism has been dominated by the idea of biochemical unity, i.e., that the central reaction sequences in metabolism are universally conserved between all forms of life. However, biochemical research in the last decades has revealed a surprising diversity in the central carbon metabolism of different microorganisms. Here, we will embrace this biochemical diversity and explain how genetic redundancy and functional degeneracy cause the diversity observed in central metabolic pathways, such as glycolysis, autotrophic CO2 fixation, and acetyl-CoA assimilation. We conclude that this diversity is not the exception, but rather the standard in microbiology.

A catalogue of biochemically diverse CRISPR-Cas9 orthologs

Bacterial Cas9 nucleases from type II CRISPR-Cas antiviral defence systems have been repurposed as genome editing tools. Although these proteins are found in many microbes, only a handful of variants are used for these applications. Here, we use bioinformatic and biochemical analyses to explore this largely uncharacterized diversity. We apply cell-free biochemical screens to assess the protospacer adjacent motif (PAM) and guide RNA (gRNA) requirements of 79 Cas9 proteins, thus identifying at least 7 distinct gRNA classes and 50 different PAM sequence requirements. PAM recognition spans the entire spectrum of T-, A-, C-, and G-rich nucleotides, from single nucleotide recognition to sequence strings longer than 4 nucleotides. Characterization of a subset of Cas9 orthologs using purified components reveals additional biochemical diversity, including both narrow and broad ranges of temperature dependence, staggered-end DNA target cleavage, and a requirement for long stretches of homology between gRNA and DNA target. Our results expand the available toolset of RNA-programmable CRISPR-associated nucleases.

Cryptic or Silent? The Known Unknowns, Unknown Knowns, and Unknown Unknowns of Secondary Metabolism

ABSTRACT Microbial natural products, particularly those produced by filamentous Actinobacteria, underpin the majority of clinically used antibiotics. Unfortunately, only a few new antibiotic classes have been discovered since the 1970s, which has exacerbated fears of a postapocalyptic world in which antibiotics have lost their utility. Excitingly, the genome sequencing revolution painted an entirely new picture, one in which an average strain of filamentous Actinobacteria harbors 20 to 50 natural product biosynthetic pathways but expresses very few of these under laboratory conditions. Development of methodology to access this “hidden” biochemical diversity has the potential to usher in a second Golden Era of antibiotic discovery. The proliferation of genomic data has led to inconsistent use of “cryptic” and “silent” when referring to biosynthetic gene clusters identified by bioinformatic analysis. In this Perspective, we discuss this issue and propose to formalize the use of this terminology.