Dynamic control systems that mimic natural regulation of catabolic pathways enable rapid production of lignocellulose-derived bioproducts

Abstract

ABSTRACTExpanding the catabolic repertoire of engineered microbial bioproduction hosts enables more complete use of complex feedstocks such as lignocellulosic hydrolysates and deconstructed mixed plastics, but the deleterious effects of existing expression systems limit the maximum carry capacity for heterologous catabolic pathways. Here, we demonstrate use of a conditionally beneficial oxidative xylose catabolic pathway to improve performance of a Pseudomonas putida strain that has been engineered for growth-coupled bioconversion of glucose into the valuable bioproduct cis,cis-muconic acid. In the presence of xylose, the pathway enhances growth rate, and therefore productivity, by >60%, but the metabolic burden of constitutive pathway expression reduces growth rate by >20% in the absence of xylose. To mitigate this growth defect, we develop a xylose biosensor based on the XylR transcription factor from Caulobacter crescentus NA1000 to autonomously regulate pathway expression. We generate a library of engineered xylose-responsive promoters that cover a three order-of-magnitude range of expression levels to tune pathway expression. Using structural modeling to guide mutations, we engineer XylR with two and three orders-of-magnitude reduced sensitivity to xylose and L-arabinose, respectively. A previously developed heterologous xylose isomerase pathway is placed under control of the biosensor, which improves the growth rate with xylose as a carbon source by 10% over the original constitutively expressed pathway. Finally, the oxidative xylose catabolic pathway is placed under control of the biosensor, enabling the bioproduction strain to maintain the increased growth rate in the presence of xylose, without the growth defect incurred from constitutive pathway expression in the absence of xylose. Utilizing biosensors to autonomously regulate conditionally beneficial catabolic pathways is generalizable approach that will be critical for engineering bioproduction hosts bacteria with the wide range of catabolic pathways required for bioconversion of complex feedstocks.