Engineering a synthetic energy-efficient formaldehyde assimilation cycle in Escherichia coli.

One-carbon (C1) substrates, such as methanol or formate, are attractive feedstocks for circular bioeconomy. These substrates are typically converted into formaldehyde, serving as the entry point into metabolism. Here, we design an erythrulose monophosphate (EuMP) cycle for formaldehyde assimilation, leveraging a promiscuous dihydroxyacetone phosphate dependent aldolase as key enzyme. In silico modeling reveals that the cycle is highly energy-efficient, holding the potential for high bioproduct yields. Dissecting the EuMP into four modules, we use a stepwise strategy to demonstrate in vivo feasibility of the modules in E. coli sensor strains with sarcosine as formaldehyde source. From adaptive laboratory evolution for module integration, we identify key mutations enabling the accommodation of the EuMP reactions with endogenous metabolism. Overall, our study demonstrates the proof-of-concept for a highly efficient, new-to-nature formaldehyde assimilation pathway, opening a way for the development of a methylotrophic platform for a C1-fueled bioeconomy in the future.

Synthetic Biology Pathway to Nucleoside Triphosphates for Expanded Genetic Alphabets.

One horizon in synthetic biology seeks alternative forms of DNA that store, transcribe, and support the evolution of biological information. Here, hydrogen bond donor and acceptor groups are rearranged within a Watson-Crick geometry to get 12 nucleotides that form 6 independently replicating pairs. Such artificially expanded genetic information systems (AEGIS) support Darwinian evolution in vitro. To move AEGIS into living cells, metabolic pathways are next required to make AEGIS triphosphates economically from their nucleosides, eliminating the need to feed these expensive compounds in growth media. We report that "polyphosphate kinases" can be recruited for such pathways, working with natural diphosphate kinases and engineered nucleoside kinases. This pathway in vitro makes AEGIS triphosphates, including third-generation triphosphates having improved ability to survive in living bacterial cells. In α-32P-labeled forms, produced here for the first time, they were used to study DNA polymerases, finding cases where third-generation AEGIS triphosphates perform better with natural enzymes than second-generation AEGIS triphosphates.

Enzymatic Formation of an Artificial Base Pair Using a Modified Purine Nucleoside Triphosphate.

The expansion of the genetic alphabet with additional, unnatural base pairs (UBPs) is an important and long-standing goal in synthetic biology. Nucleotides acting as ligands for the coordination of metal cations have advanced as promising candidates for such an expansion of the genetic alphabet. However, the inclusion of artificial metal base pairs in nucleic acids mainly relies on solid-phase synthesis approaches, and very little is known about polymerase-mediated synthesis. Herein, we report the selective and high yielding enzymatic construction of a silver-mediated base pair (dImC-AgI-dPurP) as well as a two-step protocol for the synthesis of DNA duplexes containing such an artificial metal base pair. Guided by DFT calculations, we also shed light into the mechanism of formation of this artificial base pair as well as into the structural and energetic preferences. The enzymatic synthesis of the dImC-AgI-dPurP artificial metal base pair provides valuable insights for the design of future, more potent systems aiming at expanding the genetic alphabet.

An optimized methanol assimilation pathway relying on promiscuous formaldehyde-condensing aldolases in E. coli.

Engineering biotechnological microorganisms to use methanol as a feedstock for bioproduction is a major goal for the synthetic metabolism community. Here, we aim to redesign the natural serine cycle for implementation in E. coli. We propose the homoserine cycle, relying on two promiscuous formaldehyde aldolase reactions, as a superior pathway design. The homoserine cycle is expected to outperform the serine cycle and its variants with respect to biomass yield, thermodynamic favorability, and integration with host endogenous metabolism. Even as compared to the RuMP cycle, the most efficient naturally occurring methanol assimilation route, the homoserine cycle is expected to support higher yields of a wide array of products. We test the in vivo feasibility of the homoserine cycle by constructing several E. coli gene deletion strains whose growth is coupled to the activity of different pathway segments. Using this approach, we demonstrate that all required promiscuous enzymes are active enough to enable growth of the auxotrophic strains. Our findings thus identify a novel metabolic solution that opens the way to an optimized methylotrophic platform.

Use of ß3-methionine as an amino acid substrate of Escherichia coli methionyl-tRNA synthetase.

Polypeptides containing ß-amino acids are attractive tools for the design of novel proteins having unique properties of medical or industrial interest. Incorporation of ß-amino acids in vivo requires the development of efficient aminoacyl-tRNA synthetases specific of these non-canonical amino acids. Here, we have performed a detailed structural and biochemical study of the recognition and use of ß3-Met by Escherichia coli methionyl-tRNA synthetase (MetRS). We show that MetRS binds ß3-Met with a 24-fold lower affinity but catalyzes the esterification of the non-canonical amino acid onto tRNA with a rate lowered by three orders of magnitude. Accurate measurements of the catalytic parameters required careful consideration of the presence of contaminating α-Met in ß3-Met commercial samples. The 1.45 Å crystal structure of the MetRS: ß3-Met complex shows that ß3-Met binds the enzyme essentially like α-Met, but the carboxylate moiety is mobile and not adequately positioned to react with ATP for aminoacyl adenylate formation. This study provides structural and biochemical bases for engineering MetRS with improved ß3-Met aminoacylation capabilities.

Overcoming the membrane barrier: Recruitment of γ-glutamyl transferase for intracellular release of metabolic cargo from peptide vectors.

Semipermeable membranes of cells frequently pose an obstacle in metabolic engineering by limiting uptake of substrates, intermediates, or xenobiotics. Previous attempts to overcome this barrier relied on the promiscuous nature of peptide transport systems, but often suffered from low versatility or chemical instability. Here, we present an alternative strategy to transport cargo molecules across the inner membrane of Escherichia coli based on chemical synthesis of a stable cargo-peptide vector construct, transport through the peptide import system, and efficient intracellular release of the cargo by the promiscuous enzyme γ-glutamyl transferase (GGT). Retaining the otherwise periplasmic GGT in the cytoplasm was critical for the functionality of the system, as was fine-tuning its expression in order to minimize toxic effects associated to cytoplasmic GGT expression. Given the established protocols of peptide synthesis and the flexibility of peptide transport and GGT, the system is expected to be suitable for a broad range of cargoes.

Chemical Morphing of DNA Containing Four Noncanonical Bases.

The ability of alternative nucleic acids, in which all four nucleobases are substituted, to replicate in vitro and to serve as genetic templates in vivo was evaluated. A nucleotide triphosphate set of 5-chloro-2'-deoxyuridine, 7-deaza-2'-deoxyadenosine, 5-fluoro-2'-deoxycytidine, and 7-deaza-2'deoxyguanosine successfully underwent polymerase chain reaction (PCR) amplification using templates of different lengths (57 or 525mer) and Taq or Vent (exo-) DNA polymerases as catalysts. Furthermore, a fully morphed gene encoding a dihydrofolate reductase was generated by PCR using these fully substituted nucleotides and was shown to transform and confer trimethoprim resistance to E. coli. These results demonstrated that fully modified templates were accurately read by the bacterial replication machinery and provide the first example of a long fully modified DNA molecule being functional in vivo.

Base pairing involving artificial bases in vitro and in vivo.

Herein we report the synthesis of N8-glycosylated 8-aza-deoxyguanosine (N8-8-aza-dG) and 8-aza-9-deaza-deoxyguanosine (N8-8-aza-9-deaza-dG) nucleotides and their base pairing properties with 5-methyl-isocytosine (d-isoCMe), 8-amino-deoxyinosine (8-NH2-dI), 1-N-methyl-8-amino-deoxyinosine (1-Me-8-NH2-dI), 7,8-dihydro-8-oxo-deoxyinosine (8-Oxo-dI), 7,8-dihydro-8-oxo-deoxyadenosine (8-Oxo-dA), and 7,8-dihydro-8-oxo-deoxyguanosine (8-Oxo-dG), in comparison with the d-isoCMe:d-isoG artificial genetic system. As demonstrated by Tm measurements, the N8-8-aza-dG:d-isoCMe base pair formed less stable duplexes as the C:G and d-isoCMe:d-isoG pairs. Incorporation of 8-NH2-dI versus the N8-8-aza-dG nucleoside resulted in a greater reduction in Tm stability, compared to d-isoCMe:d-isoG. Insertion of the methyl group at the N1 position of 8-NH2-dI did not affect duplex stability with N8-8-aza-dG, thus suggesting that the base paring takes place through Hoogsteen base pairing. The cellular interpretation of the nucleosides was studied, whereby a lack of recognition or mispairing of the incorporated nucleotides with the canonical DNA bases indicated the extent of orthogonality in vivo. The most biologically orthogonal nucleosides identified included the 8-amino-deoxyinosines (1-Me-8-NH2-dI and 8-NH2-dI) and N8-8-aza-9-deaza-dG. The 8-oxo modifications mimic oxidative damage ahead of cancer development, and the impact of the MutM mediated recognition of these 8-oxo-deoxynucleosides was studied, finding no significant impact in their in vivo assay.

Paralogous metabolism: S-alkyl-cysteine degradation in Bacillus subtilis.

Metabolism is prone to produce analogs of essential building blocks in the cell (here named paralogous metabolism). The variants result from lack of absolute accuracy in enzyme-templated reactions as well as from molecular aging. If variants were left to accumulate, the earth would be covered by chemical waste. The way bacteria cope with this situation is essentially unexplored. To gain a comprehensive understanding of Bacillus subtilis sulphur paralogous metabolism, we used expression profiling with DNA arrays to investigate the changes in gene expression in the presence of S-methyl-cysteine (SMeC) and its close analog, methionine, as sole sulphur source. Altogether, more than 200 genes whose relative strength of induction was significantly different depending on the sulphur source used were identified. This allowed us to pinpoint operon ytmItcyJKLMNytmO_ytnIJ_rbfK_ytnLM as controlling the pathway cycling SMeC directly to cysteine, without requiring sulphur oxygenation. Combining genetic and physiological experiments, we deciphered the corresponding pathway that begins with protection of the metabolite by acetylation. Oxygenation of the methyl group then follows, and after deprotection (deacetylation), N-formyl cysteine is produced. This molecule is deformylated by the second deformylase present in B. subtilis DefB, yielding cysteine. This pathway appears to be present in plant-associated microbes.

3-Phosphono-L-alanine as pyrophosphate mimic for DNA synthesis using HIV-1 reverse transcriptase.

A series of sulf(on)ate and phosph(on)ate amino acid phosphoramidate analogues of deoxynucleotides were synthesized as potential substrates for HIV-1 reverse transcriptase. Taurine, L-cysteic acid, 3-phosphono-L-alanine, O-sulfonato-L-serine, and O-phospho-L-serine were investigated as leaving groups in an enzyme catalyzed DNA synthesis protocol. Among these analogues, the phosphonate congener performed best and 3-phosphono-L-alanine can be considered as an excellent mimic of the pyrophosphate (PPi) moiety of deoxyadenosine triphosphate, to be used in enzymatic synthesis of nucleic acids. During a single nucleotide incorporation assay the use of 3-phosphono-L-Ala-dAMP as substrate resulted in 95% conversion to a P + 1 strand in 60 min at 50 µM (a concentration 10 times less than found for L-Asp-dAMP) and with improved incorporation kinetics and less stalling. For the sequences investigated, the efficiency of the incorporation is base dependent and decreases in the order (A ≥ T = G > C). In all cases, the incorporation follows Watson-Crick rules.

Iminodiacetic-phosphoramidates as metabolic prototypes for diversifying nucleic acid polymerization in vivo.

Previous studies in our laboratory proved that certain functional groups are able to mimic the pyrophosphate moiety and act as leaving groups in the enzymatic polymerization of deoxyribonucleic acids by HIV-1 reverse transcriptase. When the potential leaving group possesses two carboxylic acid moieties linked to the nucleoside via a phosphoramidate bond, it is efficiently recognized by this error-prone enzyme, resulting in nucleotide incorporation into DNA. Here, we present a new efficient alternative leaving group, iminodiacetic acid, which displays enhanced kinetics and an enhanced elongation capacity compared to previous results obtained with amino acid deoxyadenosine phosphoramidates. Iminodiacetic acid phosphoramidate of deoxyadenosine monophosphate (IDA-dAMP) is processed by HIV-1 RT as a substrate for single nucleotide incorporation and displays a typical Michaelis-Menten kinetic profile. This novel substrate also proved to be successful in primer strand elongation of a seven-base template overhang. Modelling of this new substrate in the active site of the enzyme revealed that the interactions formed between the triphosphate moiety, magnesium ions and enzyme's residues could be different from those of the natural triphosphate substrate and is likely to involve additional amino acid residues. Preliminary testing for a potential metabolic accessibility lets us to envision its possible use in an orthogonal system for nucleic acid synthesis that would not influence or be influenced by genetic information from the outside.