Constructing de novo biosynthetic pathways for chemical synthesis inside living cells

Abstract

Living organisms have evolved a vast array of catalytic functions that make them ideally suited for the production of medicinally and industrially relevant small-molecule targets. Indeed, native metabolic pathways in microbial hosts have long been exploited and optimized for the scalable production of both fine and commodity chemicals. Our increasing capacity for DNA sequencing and synthesis has revealed the molecular basis for the biosynthesis of a variety of complex and useful metabolites and allows the de novo construction of novel metabolic pathways for the production of new and exotic molecular targets in genetically tractable microbes. However, the development of commercially viable processes for these engineered pathways is currently limited by our ability to quickly identify or engineer enzymes with the correct reaction and substrate selectivity as well as the speed by which metabolic bottlenecks can be determined and corrected. Efforts to understand the relationship among sequence, structure, and function in the basic biochemical sciences can advance these goals for synthetic biology applications while also serving as an experimental platform for elucidating the in vivo specificity and function of enzymes and reconstituting complex biochemical traits for study in a living model organism. Furthermore, the continuing discovery of natural mechanisms for the regulation of metabolic pathways has revealed new principles for the design of high-flux pathways with minimized metabolic burden and has inspired the development of new tools and approaches to engineering synthetic pathways in microbial hosts for chemical production.

Figure 1

Pipeline for construction of a de novo metabolic pathway. Enzymes from environmental hosts are identified, assembled, and transplanted into a heterologous host for target molecule production. Bottlenecks that decrease product titer are identified by a combination of in vivo and in vitro characterization to insight into their source. Incorporation of additional metabolic and regulatory elements are used to alleviate these bottlenecks, which reveals new factors that limit production yields for further optimization.

Figure 2

Chemical phenotypes of interest for de novo metabolic pathway construction.

Figure 3

Specialized structural motifs and unusual functional groups in natural products. Structural motifs and functional groups of interested are highlighted in red. (A) Unique functional groups. (B) Unusual bond couplings. (C) Strained ring structures.

Figure 4

Methods for functional gene annotation. (A) Multiple sequence alignments are often sufficient to classify an uncharacterized protein into a superfamily or family, limiting the scope of possible reactions. (B) In silico docking utilizes structural information about the protein of interest for docking potential substrates and ranking them according to favorability of binding. The results limit the size of libraries that must be screened in vitro to determine enzyme function. (C) Functional genomic approaches including protein-protein interaction screens, genetic interaction mapping, and microarray analysis facilitate gene annotation based on biological rather than biochemical function.

Figure 5

The neutral drift mechanism of enzyme evolution. (A) A sequence encoding an enzyme with a specific substrate accumulates mutations under selective pressure to maintain the original activity, resulting in the emergence of promiscuous activities and the maintenance of the initial activity. (B) When a selective pressure favoring the promiscuous activity arises, the accumulation of a small number of mutations enhances the promiscuous activity. (C) Phosphotriesterases are proposed to have evolved recently from lactonases. (D) Tetrachlorohydroquinone dehalogenase is proposed to have evolved from maleylacetoacetate isomerase (GSH, glutathione; GSSG, glutathione disulfide).

Figure 6

Engineering pathway balance. (A) Expression of pathways genes appropriate levels can be achieved by adding RNA regulatory elements. (B) Control of ribosome binding site accessibility or (C) ribosome binding site optimization can be used to tune protein expression at the translational level. (D) Variation of promoter strength or inducer concentration can be used to tune protein expression at the transcriptional level.

Figure 7

Spatial organization in natural enzyme systems. (A) Carbamoyl phosphate synthetase utilizes a physical channel that protects the labile intermediates ammonia, carboxy phosphate, and carbamate from the cellular environment. (B) In the fungal shikimate pathway, a pentafunctional polypeptide is utilized to catalyze the five reactions required to convert 3-deoxy-D-arabino-heptulosonate 7-phosphate to 5-enolpyruvylshikimate 3-phosphate. (C) The breakdown of cellulose requires multiple enzymes of the glycosyl hydrolase superfamily, including reducing and non-reducing end exoglucanases as well as endoglucanases. The spatial organization of cellulosomes allows the modular docking and exchange of different enzymes in a single scaffold for synergistic and tunable degradation of the sugar polymer (CBM, celluolose binding module). (D) Formation of the purinosome complex between the enzymes that catalyze de novo purine biosynthesis is believed to protect the short-lived intermediates, phosphoribosylamine (PRA) and 4-carboxyaminoimidazole ribonucleotide (CAIR), in the mammalian pathway between 5-phosphoribosyl-α-pyrophosphate (PRPP) and inosine monophosphate (IMP). In most bacteria, yeasts, and plants, the precursor to CAIR, N⁵-carboxy-4-aminoimidazole ribonucleotide (N⁵-CAIR, t_½ = 15 s), also exists as a short-lived intermediate but is channeled by a multifunctional enzyme in mammals.