Biocatalytic Production of 1,2,4-Butanetriol beyond a Titer of 100 g/L: Boosting by Intermediates

Many platform chemicals such as 1,2,4-butanetriol (BTO) are still derived from petrochemicals. BTO as a versatile compound with applications ranging from pharmaceutical synthons to plasticizers is of great interest to be manufactured from renewable resources. Albeit biosynthetic pathways to produce BTO from pentoses were proposed two decades ago, no studies have reported production at high yields and titers typically demanded by chemical industries. In this work, we aimed to tackle this challenge by combining several strategies. Selection of suitable enzymes and reaction optimization in a 500 μL lab scale allowed us to achieve a titer of 1.2 M (125 g/L) (S)-BTO from 180 g/L d-xylose with a yield >97%. By the addition of an intermediate of the cascade, we could reduce up to 90% of the original redox cofactor used while still maintaining a space-time yield (STY) of 3.7 g/L/h. By applying the same approach, which we term "intermediate boosting", we could push the STY to 9.4 g/L/h. After having identified byproduct formation as a possible bottleneck, we increased production of (S)-BTO further to 1.6 M (170 g/L), approaching the toxicity level of BTO at 200 g/L that microorganisms can handle. We demonstrated, however, that our enzymes were still functional at 300 g/L BTO. Finally, we proposed several strategies to further increase the titer and yield of BTO as a feasible alternative to the petroleum-based synthetic route. This work highlights the importance of a combinatorial approach to boost the enzymatic biosynthesis of chemicals.