Engineering Terpene Production Pathways in Methylobacterium extorquens AM1.

Terpenes are diverse specialized metabolites naturally found within plants and have important roles in inter-species communication, adaptation and interaction with the environment. Their industrial applications span a broad range, including fragrances, flavors, cosmetics, natural colorants to agrochemicals and therapeutics, yet formal chemical synthesis is economically challenging due to structural complexities. Engineering terpene biosynthesis could represent an alternative in microbial biotechnological workhorses, such as Saccharomyces cerevisiae or Escherichi coli, utilizing sugars or complex media as feedstocks. Host species that metabolize renewable and affordable carbon sources may offer unique sustainable biotechnological alternatives. Methylotrophs are bacteria with the capacity to utilize one-carbon feedstocks, such as methanol or formate. They colonize the phyllosphere (above-ground area) of plants, and many accumulate abundant carotenoid pigments. Methylotrophs have the capacity to take up and use a subset of the rare earth elements known as lanthanides. These metals can enhance one-carbon (methylotrophic) metabolism. Here, we investigated whether manipulating the metabolism enables and enhances terpene production. A carotenoid-deficient mutant potentially liberates carbon, which may contribute to bioproduct accumulation. To test this hypothesis, terpene-producing bacterial strains regulated by two distinct promoters were generated. Wildtype Methylobacterium extorquens, ∆Meta1_3665, a methylotrophic mutant lacking the carotenoid pathway, and an E. coli strain were transformed with an exogenous terpene pathway and grown both in the presence and absence of lanthanides. The extraction, and the comparison of analytical profiles, provided evidence that engineered cultured M. extorquens under control of a native, inducible methylotrophic promoter can yield the sesquiterpene patchoulol when supplemented with lanthanide. In contrast, using a moderate-strength constitutive promoter failed to give production. We demonstrated colonization of the phyllosphere with the engineered strains, supporting the future engineering of selected species of the plant microbiome and with promising implications for the synthetic biology of small molecules.

Scalable and Consolidated Microbial Platform for Rare Earth Element Leaching and Recovery from Waste Sources.

Chemical methods for the extraction and refinement of technologically critical rare earth elements (REEs) are energy-intensive, hazardous, and environmentally destructive. Current biobased extraction systems rely on extremophilic organisms and generate many of the same detrimental effects as chemical methodologies. The mesophilic methylotrophic bacterium Methylobacterium extorquens AM1 was previously shown to grow using electronic waste by naturally acquiring REEs to power methanol metabolism. Here we show that growth using electronic waste as a sole REE source is scalable up to 10 L with consistent metal yields without the use of harsh acids or high temperatures. The addition of organic acids increases REE leaching in a nonspecific manner. REE-specific bioleaching can be engineered through the overproduction of REE-binding ligands (called lanthanophores) and pyrroloquinoline quinone. REE bioaccumulation increases with the leachate concentration and is highly specific. REEs are stored intracellularly in polyphosphate granules, and genetic engineering to eliminate exopolyphosphatase activity increases metal accumulation, confirming the link between phosphate metabolism and biological REE use. Finally, we report the innate ability of M. extorquens to grow using other complex REE sources, including pulverized smartphones, demonstrating the flexibility and potential for use as a recovery platform for these critical metals.

Employing methylotrophs for a green economy: one-carbon to fuel them all and through metabolism redesign them.

Microbial platforms are currently being optimized to revolutionize industrial energy production while mitigating shortages of global resources and food supplies. Here, we address recent advances to develop bacterial methylotrophic platforms as promising platforms enabling the reuse of products and materials (at their highest value) while reducing waste and pollution.

Lanthanides in Methylotrophy.

Lanthanides were previously thought to be biologically inert owing to their low solubility; however, they have recently been shown to strongly impact the metabolism of methylotrophic bacteria. Leading efforts in this emergent field have demonstrated far-reaching impacts of lanthanide metabolism in biology; from the identification of novel roles of enzymes and pathways dependent on lanthanide-chemistry to the control of transcriptional regulatory networks to the modification of microbial community interactions. Even further, the recent discovery of lanthanide-dependent enzymes associated with multi-carbon metabolism in both methylotrophs and non-methylotrophs alike suggests that lanthanide biochemistry may be more widespread than initially thought. Current efforts aim to understand how lanthanide chemistry and lanthanide-dependent enzymes affect numerous ecosystems and metabolic functions. These efforts will likely have a profound impact on biotechnological processes involving methylotrophic communities and the biologically mediated recovery of these critical metals from a variety of waste streams while redefining our understanding of a fundamental set of metals in biology.

Lanthanide Chemistry: From Coordination in Chemical Complexes Shaping Our Technology to Coordination in Enzymes Shaping Bacterial Metabolism.

Lanthanide chemistry has only been extensively studied for the last 2 decades, when it was recognized that these elements have unusual chemical characteristics including fluorescent and potent magnetic properties because of their unique 4f electrons.1,2 Chemists are rapidly and efficiently integrating lanthanides into numerous compounds and materials for sophisticated applications. In fact, lanthanides are often referred to as "the seeds of technology" because they are essential for many technological devices including smartphones, computers, solar cells, batteries, wind turbines, lasers, and optical glasses.3-6 However, the effect of lanthanides on biological systems has been understudied. Although displacement of Ca2+ by lanthanides in tissues and enzymes has long been observed,7 only a few recent studies suggest a biological role for lanthanides based on their stimulatory properties toward some plants and bacteria.8,9 Also, it was not until 2011 that the first biochemical evidence for lanthanides as inherent metals in bacterial enzymes was published.10 This forum provides an overview of the classical and current aspects of lanthanide coordination chemistry employed in the development of technology along with the biological role of lanthanides in alcohol oxidation. The construction of lanthanide-organic frameworks will be described. Examples of how the luminescence field is rapidly evolving as more information about lanthanide-metal emissions is obtained will be highlighted, including biological imaging and telecommunications.11 Recent breakthroughs and observations from different exciting areas linked to the coordination chemistry of lanthanides that will be mentioned in this forum include the synthesis of (i) macrocyclic ligands, (ii) antenna molecules, (iii) coordination polymers, particularly nanoparticles, (iv) hybrid materials, and (v) lanthanide fuel cells. Further, the role of lanthanides in bacterial metabolism will be discussed, highlighting the discovery that lanthanides are cofactors in biology, particularly in the enzymatic oxidation of alcohols. Finally, new and developing chemical and biological lanthanide mining and recycling extraction processes will be introduced.

Large-Effect Beneficial Synonymous Mutations Mediate Rapid and Parallel Adaptation in a Bacterium.

Contrary to previous understanding, recent evidence indicates that synonymous codon changes may sometimes face strong selection. However, it remains difficult to generalize the nature, strength, and mechanism(s) of such selection. Previously, we showed that synonymous variants of a key enzyme-coding gene (fae) of Methylobacterium extorquens AM1 decreased enzyme production and reduced fitness dramatically. We now show that during laboratory evolution, these variants rapidly regained fitness via parallel yet variant-specific, highly beneficial point mutations in the N-terminal region of fae These mutations (including four synonymous mutations) had weak but consistently positive impacts on transcript levels, enzyme production, or enzyme activity. However, none of the proposed mechanisms (including internal ribosome pause sites or mRNA structure) predicted the fitness impact of evolved or additional, engineered point mutations. This study shows that synonymous mutations can be fixed through strong positive selection, but the mechanism for their benefit varies depending on the local sequence context.