A combined experimental and modelling approach for the Weimberg pathway optimisation

Abstract

The oxidative Weimberg pathway for the five-step pentose degradation to α-ketoglutarate is a key route for sustainable bioconversion of lignocellulosic biomass to added-value products and biofuels. The oxidative pathway from Caulobacter crescentus has been employed in in-vivo metabolic engineering with intact cells and in in-vitro enzyme cascades. The performance of such engineering approaches is often hampered by systems complexity, caused by non-linear kinetics and allosteric regulatory mechanisms. Here we report an iterative approach to construct and validate a quantitative model for the Weimberg pathway. Two sensitive points in pathway performance have been identified as follows: (1) product inhibition of the dehydrogenases (particularly in the absence of an efficient NAD + recycling mechanism) and (2) balancing the activities of the dehydratases. The resulting model is utilized to design enzyme cascades for optimized conversion and to analyse pathway performance in C. cresensus cellfree extracts.

Characterization of highly active 2-keto-3-deoxy-L-arabinonate and 2-keto-3-deoxy-D-xylonate dehydratases in terms of the biotransformation of hemicellulose sugars to chemicals

“…S 1 ) as compared to Cc D-KdpD1, could suggest that the N-terminus for D-KdpD is important to enzyme activity and/or stability, and that the addition of further amino acids N-terminally would impede either. However, in a recent study, N-terminally fused His-tag of Cc KdpD2 (bearing additional 20 amino acids in N-terminus in comparison to C-histag of Cc KdpD2 in this study) showed a magnitude of activity similar to Cc KdpD2 (Shen et al 2020 ). Further analysis using BLAST searches in the NCBI and UniProt database suggest that the previously cloned and characterized Cc D-KdpD1 might be cloned with the wrong start codon (Tai et al 2016 ), since most of the homologs do not show the 58 amino acids extension at the N-terminus.…”

“…The authors (as well as this study) have demonstrated a higher catalytic efficiency for Px D-KdpD than for Cc D-KdpD1 (Table 2 ). Furthermore, in the recent study combining enzymatic and modeling approach, the authors were able to predict and simulate the activity of all five Weimberg enzymes, including Cc D-KdpD2 in cell-free extracts based on their kinetic model (Shen et al 2020 ).…”

“…1 ), D-xylonate could accumulate to this relevant range. The [Fe-S]-dependent sugar acid dehydratase has been reported as being one of the major bottlenecks in in vitro and in vivo utilization of the Weimberg pathway (Salusjärvi et al 2017 ; Boer et al 2019 ; Shen et al 2020 ). Therefore, a more active D-xylonate dehydratase would be needed to avoid an accumulation of D-xylonate that would inhibit Pp D-KdpD.…”

Optimization of 1,2,4‐butanetriol production from xylose in Saccharomyces cerevisiae by metabolic engineering of NADH/NADPH balance

“…xylose. BT synthesis from xylose is more attractive than BT synthesis from glucose-derived malate because of the 100% theoretical yield of the former xylose pathway (Li, Cai, Li, & Zhang, 2015;Shen et al, 2020;Z. Zhao, Xian, Liu, & Zhao, 2020).…”

SynPharm and the guide to pharmacology database: A toolset for conferring drug control on engineered proteins

“…Broadly, the challenge of optimizing engineered biological systems increases exponentially with the number of components involved. [1][2][3] This challenge does not lie primarily in physical construction of DNA sequences and so forth, because any difficulties here increase only linearly with system size. Rather, it arises because each extra active molecule in a system adds further dimensions to the parameter space in which the finished system will operate.…”