ß-Dicarbonyls Facilitate Engineered Microbial Bromoform Biosynthesis.

Ruminant livestock produce around 24% of global anthropogenic methane emissions. Methanogenesis in the animal rumen is significantly inhibited by bromoform, which is abundant in seaweeds of the genus Asparagopsis. This has prompted the development of livestock feed additives based on Asparagopsis to mitigate methane emissions, although this approach alone is unlikely to satisfy global demand. Here we engineer a non-native biosynthesis pathway to produce bromoform in vivo with yeast as an alternative biological source that may enable sustainable, scalable production of bromoform by fermentation. ß-dicarbonyl compounds with low pKa values were identified as essential substrates for bromoform production and enabled bromoform synthesis in engineered Saccharomyces cerevisiae expressing a vanadate-dependent haloperoxidase gene. In addition to providing a potential route to the sustainable biological production of bromoform at scale, this work advances the development of novel microbial biosynthetic pathways for halogenation.

A Simple Recombinant E. coli Cell Lysate-Based Biocatalyst for ATP-Dependent Multi-step Reactions.

The utility of ATP-dependent multi-enzymatic reactions is limited by their requirement for stoichiometric amounts of this expensive cofactor or additional purified enzymes for its recycling. Here we describe a simple method for the production of recombinant cell-free extracts (or lysates) of E. coli that support ATP-dependent biotransformations. The inexpensive preparation described is obtained with modest processing from a single recombinant bacterial culture of E. coli. In addition to recombinantly overexpressed enzymes that catalyze the primary ATP-dependent reactions of interest, endogenous kinases that are naturally present in the extract catalyze recycling of the requisite ATP. This means that only catalytic amounts of cofactor are necessary to drive the biotransformation, and without the requirement for additional purified enzymes. This approach has been applied successfully to an array of in vitro enzymatic cascades with multiple ATP-dependent steps.

Recombinant cell-lysate-catalysed synthesis of uridine-5'-triphosphate from nucleobase and ribose, and without addition of ATP.

Nucleoside triphosphates (NTPs) are important synthetic targets with diverse applications in therapeutics and diagnostics. Enzymatic routes to NTPs from simple building blocks are attractive, however the cost and complexity of assembling the requisite mixtures of multiple enzymes hinders application. Here, we describe the use of an engineered E. coli cell-free lysate as an efficient readily-prepared multi-enzyme biocatalyst for the production of uridine triphosphate (UTP) from free ribose and nucleobase. Endogenous lysate enzymes are able to support the nucleobase ribosylation and nucleotide phosphorylation steps, while uridine phosphorylation and the production of ribose phosphates (ribose 1-phosphate, ribose 5-phosphate and phosphoribosyl pyrophosphate) require recombinant enrichment of endogenous activities. Co-expression vectors encoding all required recombinant enzymes were employed for host cell transformation, such that a cell-free lysate with all necessary activities was obtained from a single bacterial culture. ATP required as phosphorylation cofactor was recycled by endogenous lysate enzymes using cheap, readily-prepared acetyl phosphate. Surprisingly, acetyl phosphate initiated spontaneous generation of ATP in the lysate, most likely from the breakdown of endogenous pools of adenosine-containing starting materials (e.g. adenosine cofactors, ribonucleic acids). The sub-stoichiometric amount of ATP produced and recycled in this way was enough to support all ATP-dependent steps without addition of any exogenous cofactor or auxiliary enzyme. Using this approach, equimolar solutions of orotic acid and ribose are transformed near quantitatively into 1.4 g L-1 UTP within 2.5 h, using a low-cost, readily-generated biocatalytic preparation.

DNA amplification with in situ nucleoside to dNTP synthesis, using a single recombinant cell lysate of E. coli.

Nucleic acid amplification (NAA) is a cornerstone of modern molecular and synthetic biology. Routine application by non-specialists, however, is hampered by difficulties with storing and handling the requisite labile and expensive reagents, such as deoxynucleoside triphosphates (dNTPs) and polymerases, and the complexity of protocols for their use. Here, a recombinant E. coli extract is reported that provides all the enzymes to support high-fidelity DNA amplification, and with labile dNTPs generated in situ from cheap and stable deoxynucleosides. Importantly, this is obtained from a single, engineered cell strain, through minimal processing, as a lysate capable of replacing the cold-stored commercial reagents in a typical PCR. This inexpensive preparation is highly active, as 1 L of bacterial culture is enough to supply ~106 NAA reactions. Lyophilized lysate can be used after a single-step reconstitution, resulting overall in a greatly simplified workflow and a promising synthetic biology tool, in particular for applications such as diagnostics.

ATP Recycling with Cell Lysate for Enzyme-Catalyzed Chemical Synthesis, Protein Expression and PCR.

E. coli lysate efficiently catalyzes acetyl phosphate-driven ATP regeneration in several important biotechnological applications. The utility of this ATP recycling strategy in enzyme-catalyzed chemical synthesis is illustrated through the conversion of uridine to UMP by the lysate from recombinant overexpression of uridine kinase with the E. coli. The UMP is further transformed into UTP through sequential phosphorylations by kinases naturally present in the lysate, in high yield. Cytidine and 5-fluorouridine also give the corresponding NMPs and NTPs with this system. Cell-free protein expression with a processed extract of lysate also proceeds readily when, instead of adding the required NTPs, all four are produced in situ from the NMPs, using acetyl phosphate and relying on endogenous kinase activity. Similarly, dNMPs can be used to produce the dNTPs necessary for DNA synthesis in PCR. These cheap alternative protocols showcase the potential of acetyl phosphate and ATP recycling with readily available cell lysate.