Engineering enzymes for new C1 converting pathways

Carbon fixation is one of the most important processes on Earth. Most of the CO2 in the atmosphere is fixed by oxygenic photosynthesis, using the enzyme Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). However, Rubisco suffers from side reactivity with oxygen, which ultimately accounts for biomass losses of 20-40%. Synthetic biology can offer solutions via human-designed pathways to overcome the problems of natural carbon fixation. These pathways are fast and efficient and can generally be divided into two categories: carboxylation-first pathways and reduction-first pathways. In the first, CO2 is directly assimilated, whereas in the latter, reduced C1 compounds such as formate or methanol are assimilated. Often, these pathways rely on reactions that do not exist yet, so-called new-to-nature reactions. Enzymes did not evolve to catalyze certain reactions, limiting the chemical space available to cells which is why enzyme engineering can be employed to overcome these limitations. During enzyme engineering campaigns, often a promiscuous activity is chosen that is similar to the desired new activity, in order to develop a new-to-nature reaction. In this thesis, two different enzymes are engineered for their application in carbon fixation. In Chapter I a Coenzyme B12 dependent mutase is engineered for the conversion of 3-hydroxypropionyl-CoA to lactyl-CoA, closing a gap in a carboxylating carbon assimilation pathway, the malonlyl-CoA oxaloacetate glyoxylate (MOG) cycle. This carbon fixation cycle was designed over 10 years ago as an alternative to the Calvin Benson Bassham cycle and was predicted to operate about twice as fast. The mutase- termed lactyl-CoA mutase (LCM)- has been evolved from a 2-hydroxyisobutyryl-CoA mutase from Bacillus massiliosenegalensis. The LCM module, a short pathway for the conversion of acetyl-CoA to lactyl-CoA, has been demonstrated in vitro. The enzyme was engineered using a combination of in vivo mutagenesis and growth-coupled selection to achieve a 10-fold overall improvement of the kinetic parameters of the enzyme. In Chapter II, a formic acid reductase is engineered for application in a reduction-first carbon assimilation pathway. Here, a carboxylic acid reductase from Mycobacteroides abscessus (MAB4714) is engineered into a formate reductase. Formate can now be reduced to a more reactive aldehyde, formaldehyde, which can be efficiently assimilated by cells. The carboxylic acid reductase was evolved into a formic acid reductase using classical enzyme engineering strategies and showed a five-fold improvement in catalytic efficiency, as well as more than 50-fold reduction in side-reactivity with acetate.