We describe a computationally designed enzyme, formolase (FLS), which catalyzes the carboligation of three one-carbon formaldehyde molecules into one three-carbon dihydroxyacetone molecule. The existence of FLS enables the design of a new carbon fixation pathway, the formolase pathway, consisting of a small number of thermodynamically favorable chemical transformations that convert formate into a three-carbon sugar in central metabolism. The formolase pathway is predicted to use carbon more efficiently and with less backward flux than any naturally occurring one-carbon assimilation pathway. When supplemented with enzymes carrying out the other steps in the pathway, FLS converts formate into dihydroxyacetone phosphate and other central metabolites in vitro. These results demonstrate how modern protein engineering and design tools can facilitate the construction of a completely new biosynthetic pathway.computational protein design | pathway engineering | carbon fixation N ovel strategies are needed to address current challenges in energy storage and carbon sequestration. One approach is to engineer biological systems to convert one-carbon compounds into multicarbon molecules such as fuels and other high value chemicals. Many synthetic pathways to produce value-added chemicals from common feedstocks, such as glucose, have been constructed in organisms that lack one-carbon anabolic pathways, such as Escherichia coli or Saccharomyces cerevisiae (1-3); however, despite considerable effort, it has been difficult to introduce heterologous one-carbon fixing pathways into these organisms (4). Likely problems include the inherent complexity, environmental sensitivity, inefficiency, or unfavorable chemical driving force of naturally occurring one-carbon metabolic pathways (5).An optimal pathway for one-carbon utilization in common synthetic biology platforms would be (i) composed of a minimal number of enzymes, (ii) linear and disconnected from other metabolic pathways, (iii) thermodynamically favorable with a significant driving force at most or all steps, and (iv) capable of functioning in a robust manner under both aerobic and anaerobic conditions (5). A pathway with these properties could enable the assimilation of one-carbon molecules as the sole carbon source for the production of fuels and chemicals. Although no such pathway is known in nature, the established electrochemical reduction of carbon dioxide to formate under ambient temperatures and pressures in neutral aqueous solutions provides an attractive starting point for a onecarbon fixation pathway (5-8).We describe the computational design of an enzyme that catalyzes the carboligation of three one-carbon molecules into a single three-carbon molecule. This enzyme enables the construction of a new pathway, the formolase pathway, in which formate is converted into the central metabolite dihydroxyacetone phosphate (DHAP; Fig. 1). The use of computational protein design to reengineer catalytic activities opens up the pathway design space beyond that available based o...

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P. putida lysine metabolism can produce multiple commodity chemicals, conferring great biotechnological value. Despite much research, the connection of lysine catabolism to central metabolism in P. putida remained undefined. Here, we used random barcode transposon sequencing to fill the gaps of lysine metabolism in P. putida. We describe a route of 2-oxoadipate (2OA) catabolism, which utilizes DUF1338-containing protein P. putida 5260 (PP_5260) in bacteria. Despite its prevalence in many domains of life, DUF1338-containing proteins have had no known biochemical function. We demonstrate that PP_5260 is a metalloenzyme which catalyzes an unusual route of decarboxylation of 2OA to d-2-hydroxyglutarate (d-2HG). Our screen also identified a recently described novel glutarate metabolic pathway. We validate previous results and expand the understanding of glutarate hydroxylase CsiD by showing that can it use either 2OA or 2KG as a cosubstrate. Our work demonstrated that biological novelty can be rapidly identified using unbiased experimental genetics and that RB-TnSeq can be used to rapidly validate previous results.

30Despite intensive study for 50 years, the biochemical and genetic links between lysine 31 metabolism and central metabolism in Pseudomonas putida remain unresolved. To establish 32 these biochemical links, we leveraged Random Barcode Transposon Sequencing (RB-TnSeq), a 33 genome-wide assay measuring the fitness of thousands of genes in parallel, to identify multiple 34 novel enzymes in both L-and D-lysine metabolism. We first describe three pathway enzymes 35 that catabolize L-2-aminoadipate (L-2AA) to 2-ketoglutarate (2KG), connecting D-lysine to the 36 TCA cycle. One of these enzymes, PP_5260, contains a DUF1338 domain, a family with no 37 previously described biological function. Our work also identified the recently described CoA 38 independent route of L-lysine degradation that metabolizes to succinate. We expanded on 39 previous findings by demonstrating that glutarate hydroxylase CsiD is promiscuous in its 2-40 oxoacid selectivity. Proteomics of select pathway enzymes revealed that expression of catabolic 41 genes is highly sensitive to particular pathway metabolites, implying intensive local and global 42 regulation. This work demonstrates the utility of RB-TnSeq for discovering novel metabolic 43 pathways in even well-studied bacteria, as well as a powerful tool for validating previous 44 research. 45 Importance 46 P. putida lysine metabolism can produce multiple commodity chemicals, conferring great 47 biotechnological value. Despite much research, connecting lysine catabolism to central 48 metabolism in P. putida remained undefined. Herein we use Random Barcode Transposon 49Sequencing to fill in the gaps of lysine metabolism in P. putida. We describe a route of 2-50 oxoadipate (2OA) catabolism in bacteria, which utilizes DUF1338 containing protein PP_5260. 51Despite its prevalence in many domains of life, DUF1338 containing proteins had no known 52 biochemical function. We demonstrate PP_5260 is a metalloenzyme which catalyzes an unusual 53 2OA to D-2HG decarboxylation. Our screen also identified a recently described novel glutarate 54 metabolic pathway. We validate previous results, and expand the understanding of glutarate 55hydroxylase CsiD by showing can it use either 2OA or 2KG as a cosubstrate. Our work 56 demonstrates biological novelty can be rapidly identified using unbiased experimental genetics, 57and that RB-TnSeq can be used to rapidly validate previous results. 58 Introduction 59Pseudomonas putida is an ubiquitous saprophytic soil bacterium and is a model organism 60for bioremediation (1). Interest in utilizing P. putida KT2440 as a chassis organism for metabolic 61 engineering has recently surged due to the existence of well-established genetic tools and its 62 robust metabolism of aromatic compounds that resemble lignin hydrolysis products (2-4). As 63 lignin valorization remains essential for the economic feasibility of cellulosic bioproducts, a 64 nuanced and predictable understanding of P. putida metabolism is highly desirable (5). 65Although its aromatic metabolism has garnered mu...

With its ability to catabolize a wide variety of carbon sources and a growing engineering toolkit, Pseudomonas putida KT2440 is emerging as an important chassis organism for metabolic engineering. Despite advances in our understanding of this organism, many gaps remain in our knowledge of the genetic basis of its metabolic capabilities. These gaps are particularly noticeable in our understanding of both fatty acid and alcohol catabolism, where many paralogs putatively coding for similar enzymes co-exist making biochemical assignment via sequence homology difficult. To rapidly assign function to the enzymes responsible for these metabolisms, we leveraged Random Barcode Transposon Sequencing (RB-TnSeq). Global fitness analyses of transposon libraries grown on 13 fatty acids and 10 alcohols produced strong phenotypes for hundreds of genes. Fitness data from mutant pools grown on varying chain length fatty acids indicated specific enzyme substrate preferences, and enabled us to hypothesize that DUF1302/DUF1329 family proteins potentially function as esterases. From the data we also postulate catabolic routes for the two biogasoline molecules isoprenol and isopentanol, which are catabolized via leucine metabolism after initial oxidation and activation with CoA. Because fatty acids and alcohols may serve as both feedstocks or final products of metabolic engineering efforts, the fitness data presented here will help guide future genomic modifications towards higher titers, rates, and yields. IMPORTANCE To engineer novel metabolic pathways into P. putida, a comprehensive understanding of the genetic basis of its versatile metabolism is essential. Here we provide functional evidence for the putative roles of hundreds of genes involved in the fatty acid and alcohol metabolism of this bacterium. These data provide a framework facilitating precise genetic changes to prevent product degradation and channel the flux of specific pathway intermediates as desired.

Advances in retooling microorganisms have enabled bioproduction of ‘drop-in’ biofuels, fuels that are compatible with existing spark-ignition, compression-ignition, and gas-turbine engines. As the majority of petroleum consumption in the United States consists of gasoline (47%), diesel fuel and heating oil (21%), and jet fuel (8%), ‘drop-in’ biofuels that replace these petrochemical sources are particularly attractive. In this review, we discuss the application of aldehyde decarbonylases to produce gasoline substitutes from fatty acid products, a recently crystallized reductase that could hydrogenate jet fuel precursors from terpene synthases, and the exquisite control of polyketide synthases to produce biofuels with desired physical properties (e.g., lower freezing points). With our increased understanding of biosynthetic logic of metabolic pathways, we discuss the unique advantages of fatty acid, terpene, and polyketide synthases for the production of bio-based gasoline, diesel and jet fuel.

Lepidoptera (butterflies and moths) make the six-carbon compounds homoisopentenyl pyrophosphate (HIPP) and homodimethylallyl pyrophosphate (HDMAPP) that are incorporated into sixteen, seventeen and eighteen carbon farnesyl pyrophosphate (FPP) analogues. In this work we heterologously expressed the lepidopteran modified mevalonate pathway, a propionyl-CoA ligase, and terpene cyclases in E. coli to produce several novel terpenes containing sixteen carbons. Changing the terpene cyclase generated different novel terpene product profiles. To further validate the new compounds we confirmed 13 C propionate was incorporated, and that the masses and fragmentation patterns were consistent with novel sixteen carbon terpenes by GC-QTOF. Based on the available farnesyl pyrophosphate analogues lepidoptera produce, this approach should greatly expand the reachable biochemical space with applications in areas where terpenes have traditionally found uses.

Past anthrax attacks in the United States have highlighted the need for improved measures against bioweapons. The virulence of anthrax stems from the shielding properties of the Bacillus anthracis poly-γ-d-glutamic acid capsule. In the presence of excess CapD, a B. anthracis γ-glutamyl transpeptidase, the protective capsule is degraded, and the immune system can successfully combat infection. Although CapD shows promise as a next generation protein therapeutic against anthrax, improvements in production, stability, and therapeutic formulation are needed. In this study, we addressed several of these problems through computational protein engineering techniques. We show that circular permutation of CapD improved production properties and dramatically increased kinetic thermostability. At 45 °C, CapD was completely inactive after 5 min, but circularly permuted CapD remained almost entirely active after 30 min. In addition, we identify an amino acid substitution that dramatically decreased transpeptidation activity but not hydrolysis. Subsequently, we show that this mutant had a diminished capsule degradation activity, suggesting that CapD catalyzes capsule degradation through a transpeptidation reaction with endogenous amino acids and peptides in serum rather than hydrolysis.