Recent advances in metabolic engineering have demonstrated that microbial biosynthesis can provide a viable alternative to chemical synthesis for the production of bulk and fine chemicals. Introduction of a new biosynthetic pathway typically requires the expression of multiple heterologous enzymes in the production host, which can impose stress on the host cell and, thereby, limit performance of the pathway. Unfortunately, analysis and treatment of the host stress response can be difficult, because there are many sources of stress that may interact in complex ways. We use a systems biological approach to analyze the stress imposed by expressing different enzyme variants from a lineage of soluble P450 monooxygenases, previously evolved for heterologous activity in Saccharomyces cerevisiae . Our analysis identifies patterns of stress imposed on the host by heterologous enzyme overexpression that are consistent across the evolutionary lineage, ultimately implicating heme depletion as the major stress. We show that the monooxygenase evolution, starting from conditions of either high or low stress, caused the cellular stress to converge to a common level. Overexpression of rate-limiting enzymes in the endogenous heme biosynthetic pathway alleviates the stress imposed by expression of the P450 monooxygenases and increases the enzymatic activity of the final evolved P450 by an additional 2.3-fold. Heme overexpression also increases the total activity of an endogenous cytosolic heme-containing catalase but not a heterologous P450 that is membrane-associated. This work demonstrates the utility of combining systems and synthetic biology to analyze and optimize heterologous enzyme expression.

Summary: The rapidly growing industry of crop biostimulants leverages the application of plant growth promoting rhizobacteria (PGPR) to promote plant growth and health. However, introducing nonnative rhizobacteria may impact other aspects of ecosystem functioning and have legacy effects; these potential consequences are largely unexplored. Nontarget consequences of PGPR may include changes in resident microbiomes, nutrient cycling, pollinator services, functioning of other herbivores, disease suppression, and organic matter persistence. Importantly, we lack knowledge of whether these ecosystem effects may manifest in adjacent ecosystems. The introduced PGPR can leave a functional legacy whether they persist in the community or not. Legacy effects include shifts in resident microbiomes and their temporal dynamics, horizontal transfer of genes from the PGPR to resident taxa, and changes in resident functional groups and interaction networks. Ecosystem functions may be affected by legacies PGPR leave following niche construction, such as when PGPR alter soil pH that in turn alters biogeochemical cycling rates. Here, we highlight new research directions to elucidate how introduced PGPR impact resident microbiomes and ecosystem functions and their capacity for legacy effects.

Transposon mutagenesis is a powerful technique in microbial genetics for the identification of genes in uncharacterized pathways. Recently, the throughput of transposon mutagenesis techniques has been dramatically increased through the combination of DNA barcoding and high-throughput sequencing. Here we show that, when applied to catabolic pathways, barcoded transposon libraries can be used to distinguish redundant pathways, decompose complex pathways into substituent modules, discriminate between enzyme homologs, and rapidly identify previously-hypothetical enzymes in an unbiased genome-scale search. We use this technique to identify two genes, which we name desC and desD, are involved in the degradation of the lignin-derived aromatic compound sinapic acid in the non-model bacterium Novosphingobium aromaticivorans. We show that DesC is a methyl-esterase acting on an intermediate formed during sinapic acid catabolism, providing the last enzyme in a proposed catabolic pathway. This approach will be particularly useful in the identification of complete pathways suitable for heterologous expression in metabolic engineering. Importance Identification of the genes involved in specific biochemical transformations is a key step in predicting microbial function from nucleic acid sequences and in engineering microbes to endow them with new functions. We have shown that new techniques for transposon mutagenesis can dramatically simplify this process and allow the rapid identification of genes in uncharacterized pathways. These techniques provide the necessary scale to fully elucidate complex biological networks such as those used to degrade mixtures of lignin-derived aromatic compounds.