It is a common misconception in whole-cell biocatalysis to refer to an enzyme as the biocatalyst, thereby neglecting the structural and metabolic framework provided by the cell. Here, the low whole-cell biocatalyst stability, that is, the stability of specific biocatalyst activity, in a process for the terminal oxyfunctionalization of renewable fatty acid methyl esters was investigated. This reaction, which is difficult to achieve by chemical means, is catalyzed by Escherichia coli featuring the monooxygenase system AlkBGT and the uptake facilitator AlkL from Pseudomonas putida GPo1. Corresponding products, that is, terminal alcohols, aldehydes, and acids, constitute versatile bifunctional building blocks, which are of special interest for polymer synthesis. It could clearly be shown that extensive dodecanoic acid methyl ester uptake mediated by high AlkL levels leads to whole-cell biocatalyst toxification. Thus, cell viability constitutes the primary factor limiting biocatalyst stability and, as a result, process durability. Hence, a compromise had to be found between low biocatalyst activity due to restricted substrate uptake and poor biocatalyst stability due to AlkL-mediated toxification. This was achieved by the fine-tuning of heterologous alkL expression, which, furthermore, enabled the identification of the alkBGT expression level as another critical factor determining biocatalyst stability. Controlled synthesis of AlkL and reduced alkBGT expression finally enabled an increase of product titers by a factor of 4.3 up to 229 g L in a two-liquid phase bioprocess setup. Clearly, ω-oxyfunctionalization process performance was determined by cell viability and thus biocatalyst stability rather than the maximally achievable specific biocatalyst activity. Biotechnol. Bioeng. 2017;114: 874-884. © 2016 Wiley Periodicals, Inc.

Thiabendazole (TBZ), is a persistent fungicide/anthelminthic and a serious environmental threat. We previously enriched a TBZ‐degrading bacterial consortium and provided first evidence for a Sphingomonas involvement in TBZ transformation. Here, using a multi‐omic approach combined with DNA‐stable isotope probing (SIP) we verified the key degrading role of Sphingomonas and identify potential microbial interactions governing consortium functioning. SIP and amplicon sequencing analysis of the heavy and light DNA fraction of cultures grown on 13C‐labelled versus 12C‐TBZ showed that 66% of the 13C‐labelled TBZ was assimilated by Sphingomonas. Metagenomic analysis retrieved 18 metagenome‐assembled genomes with the dominant belonging to Sphingomonas, Sinobacteriaceae, Bradyrhizobium, Filimonas and Hydrogenophaga. Meta‐transcriptomics/‐proteomics and non‐target mass spectrometry suggested TBZ transformation by Sphingomonas via initial cleavage by a carbazole dioxygenase (car) to thiazole‐4‐carboxamidine (terminal compound) and catechol or a cleaved benzyl ring derivative, further transformed through an ortho‐cleavage (cat) pathway. Microbial co‐occurrence and gene expression networks suggested strong interactions between Sphingomonas and a Hydrogenophaga. The latter activated its cobalamin biosynthetic pathway and Sphingomonas its cobalamin salvage pathway to satisfy its B12 auxotrophy. Our findings indicate microbial interactions aligning with the ‘black queen hypothesis’ where Sphingomonas (detoxifier, B12 recipient) and Hydrogenophaga (B12 producer, enjoying detoxification) act as both helpers and beneficiaries.

22Background: Thiabendazole (TBZ), is a benzimidazole fungicide and anthelminthic whose high 23 persistence and toxicity pose a serious environmental threat. In our quest for environmental mitigation 24 we previously isolated the first TBZ-degrading bacterial consortium and provided preliminary evidence 25 for its composition and the degrading role of a Sphingomonas. Here, we employed a multi-omic 26 approach combined with DNA-stable isotope probing (SIP) to determine the genetic make-up of the 27 key consortium members, to disentangle nutritional and metabolic interdependencies, to identify the 28 transformation pathway of TBZ and to understand the genetic network driving its transformation. 29Results: Time-series SIP in combination with amplicon sequencing analysis verified the key role of 30 Sphingomonas in TBZ degradation by assimilating over 80% of the 13 C-labelled phenyl moiety of TBZ. 31Non-target mass spectroscopy (MS) analysis showed the accumulation of thiazole-4-carboxamidine as 32 a single dead-end transformation product and no phenyl-containing derivative, in line with the phenyl 33 moiety assimilation in the SIP analysis. Time series metagenomic analysis of the consortium 34 supplemented with TBZ or succinate led to the assembly of 18 metagenome-assembled genomes 35 (MAGs) with >80% completeness, six (Sphingomonas 3X21F, γ-Proteobacterium 34A, 36Bradyrhizobiaceae 9B and Hydrogenophaga 19A, 13A, and 23F) being dominant. Meta-transcriptomic 37 and -proteomic analysis suggested that Sphingomonas mobilize a carbazole dioxygenase (car) operon 38 during the initial cleavage of TBZ to thiazole-4-carboxamidine and catechol, the latter is further 39 transformed by enzymes encoded in a catechol ortho-cleavage (cat) operon; both operons being up-40 regulated during TBZ degradation. Computational docking analysis of the terminal oxygenase 41 component of car, CarAa, showed high affinity to TBZ, comparable to carbazole, reinforcing its high 42 potency for TBZ transformation. These results suggest no interactions between consortium members in 43 TBZ transformation, performed solely by Sphingomonas. In contrast, gene expression network analysis 44 revealed strong interactions between Sphingomonas MAG 3X12F and Hydrogenophaga MAG 23F, 45with Hydrogenophaga activating its cobalamin biosynthetic pathway and Sphingomonas its cobalamin 46 salvage pathway along TBZ degradation. 47 Conclusions:Our findings suggest interactions between consortium members which align with the 48 "black queen hypothesis": Sphingomonas detoxifies TBZ, releasing consortium members by a toxicant; 49 in return for this, Hydrogenophaga 23F provides cobalamin to the auxotrophic Sphingomonas. 50 Background 54 Thiabendazole (TBZ) is a benzimidazole compound which is used as a post-harvest fungicide to control 55 fungal infestations on fruits during storage [1] and as a broad spectrum anthelminthic to control 56 endoparasites in livestock farming [2]. It acts by binding to tubulin monomers inhibiting the 57 polymerization of microtubules and, thus,...

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