Aldehyde Production in Crude Lysate- and Whole Cell-Based Biotransformation Using a Noncanonical Redox Cofactor System

Abstract

It is challenging to biosynthesize industrially important aldehydes, which are readily consumed by the numerous alcohol dehydrogenases (ADHs) in cells. In this work, we demonstrate that a nicotinamide mononucleotide (NMN + )-dependent redox cofactor cycling system enables aldehyde accumulation in Escherichia coli crude lysates and whole cells. By specifically delivering reducing power to a recombinant enoate reductase, but not to endogenous ADHs, we convert citral to citronellal with minimal byproduct formation (97−100% and 83% product purity in crude lysate-and whole cell-based biotransformation, respectively). We envision the system's universal application to lowering the noise in biomanufacturing by silencing the host's metabolic background.

LuxAB-Based Microbial Cell Factories for the Sensing, Manufacturing and Transformation of Industrial Aldehydes

“…Citronellal (16b) is not only used as a scent, but has insect repellent and antifungal properties [70,71]. Excitingly, the AlkJ-catalyzed oxidation of 15a exclusively formed 15b (>80% GC yield; Figure 6A), offering a biocatalytic alternative to chemical oxidation procedures [72] and enabling combinations with other enzymatic transformations, for example, the XenAmediated reduction of the proximal C = C double bond, yielding 16b, as suggested by Richardson and co-workers [61]. Citral (15b) is widely used in perfumes, as mosquito repellant [73] and is an important precursor for other terpenes [74] and carotenoids including vitamin A [75].…”

“…Furthermore, 16b, which lacks the proximal C=C double bond, was detected (11.0 ± 0.8%) after 24 h, (Figure 5). As before, these byproducts are produced by host enzymatic activities [9,15,20,[59][60][61].…”

“…Furthermore, 16b which lacks the proximal C=C double bond, was detected (11.0 ± 0.8%) after 24 h, (Figure 5). As before, these byproducts are produced by host enzymatic activities [9,15,20,[59][60][61] S11. Ex periments were performed in RCs of E. coli RARE (OD600 ≈ 10.0) expressing CARMm/PPTNi from pACYCDuet-1 in the presence of 5 mM CA (c) and 5% (ν/ν) ethanol as organic co-solvent.…”

Directed evolution of phosphite dehydrogenase to cycle noncanonical redox cofactors via universal growth selection platform

“…Our previous work developed nicotinamide mononucleotide (NMN + ), a truncated version of the native nicotinamide cofactors, as an efficient noncanonical redox cofactor 16 , 17 . Compared to other simpler NAD + mimetics, NMN + can be produced renewably using low-cost feedstocks through biosynthetic pathways 12 , 13 , 18 – 20 .…”

Cell-Free Total Biosynthesis of Plant Terpene Natural Products Using an Orthogonal Cofactor Regeneration System

“…In particular, efficient and orthogonal cofactor supply and regeneration is an ever-present obstacle, especially for more complex pathways in which multiple (redox) cofactors are involved. Numerous approaches have been developed to address this obstacle, including reengineering of enzyme cofactor specificity, 8 use of chemically orthogonal unnatural cofactors, 9,10 and, in an impressive demonstration, the use of a molecular rheostat and synthetic purge valve to manage excess cofactor buildup. 11,12 Indeed, balancing cofactor usage is especially important in systems where both reductive and oxidative reactions are involved.…”