Barcelos, C. A. | Search bioRxiv

bioRxiv2022

A highly active Burkholderia polyketoacyl-CoA thiolase for production of triacetic acid lactone

Triacetic acid lactone (TAL) is a platform chemical biosynthesized primarily through decarboxylative Claisen condensation by type III polyketide synthase 2-pyrone synthase (2-PS). However, this reaction suffers from intrinsic energy inefficiency and feedback inhibition by and competition for malonyl-CoA. TAL production through non-decarboxylative Claisen condensation by polyketoacyl-CoA thiolase alleviates many of these disadvantages. We discovered five more thiolases with TAL production activity by exploring homologs of a previously reported polyketoacyl-CoA thiolase, BktB, from Cupriavidus necator. Among them, the BktB homolog from Burkholderia sp. RF2-non_BP3 has [~] 30 times higher in vitro and in vivo TAL production activity and led to [~]10 times higher TAL titer than 2-PS when expressed in Escherichia coli, achieving a titer of 2.8 g/L in fed-batch fermentations. This discovery of a novel polyketoacyl-CoA thiolase with superior TAL production activity paves the way for realization of total biomanufacturing of TAL.

bioengineering↗

bioRxiv2020

Population dynamics analysis of Saccharomyces cerevisiae deletion library during fed-batch cultivation using Bar-seq

To understand the genetic basis of changes in strain physiology during industrial fermentation, and the corresponding roles these genes play in strain performance, we employed a barcoded yeast deletion library to assess genome-wide strain fitness across a simulated industrial fermentation regime. Our results demonstrate the utility of Bar-seq to assess fermentation associated stresses in yeast populations under industrial conditions. We find that mutant population diversity is maintained through multiple seed trains, enabling for large scale fermentation selective pressures to act upon the community. We identify specific deletion mutants that were enriched in all processes, independent of the cultivation conditions, which include MCK1, RIM11, MRK1, and YGK3 that encode homologues of mammalian glycogen synthase kinase 3 (GSK-3). Further, we show that significant changes in the population diversity during fed-batch cultivations reflect the presence of significant external stresses, such as the accumulation of the fermentative byproduct ethanol. The mutants that were lost during the time of most extreme population selection suggest that specific biological processes may be required to cope with these specific stresses. Overall our work highlights a promising avenue to identify genetic loci and biological stress responses required for fitness under industrial conditions.

microbiology↗