Engineering Escherichia coli for anaerobic alkane activation: Biosynthesis of (1‐methylalkyl)succinates

Abstract

In anoxic environments, microbial activation of alkanes for subsequent metabolism occurs most commonly through the addition of fumarate to a subterminal carbon, producing an alkylsuccinate. Alkylsuccinate synthases are complex, multi‐subunit enzymes that utilize a catalytic glycyl radical and require a partner, activating enzyme for hydrogen abstraction. While many genes encoding putative alkylsuccinate synthases have been identified, primarily from nitrate‐ and sulfate‐reducing bacteria, few have been characterized and none have been reported to be functionally expressed in a heterologous host. Here, we describe the functional expression of the (1‐methylalkyl)succinate synthase (Mas) system from Azoarcus sp. strain HxN1 in recombinant Escherichia coli. Mass spectrometry confirms anaerobic biosynthesis of the expected products of fumarate addition to hexane, butane, and propane. Maximum production of (1‐methylpentyl)succinate is observed when masC, masD, masE, masB, and masG are all present on the expression plasmid; omitting masC reduces production by 66% while omitting any other gene eliminates production. Meanwhile, deleting iscR (encoding the repressor of the E. coli iron–sulfur cluster operon) improves product titer, as does performing the biotransformation at reduced temperature (18°C), both suggesting alkylsuccinate biosynthesis is largely limited by functional expression of this enzyme system.

Development of an in vitro method for activation of X-succinate synthases for fumarate hydroalkylation

“…Even less is known about these enzymes that can directly and selectively functionalize saturated alkanes; for example, the subunit/cofactor architecture of these enzymes is still unknown and is thought to include an additional subunit. 34 Could we use a similar genome mining approach to find soluble alkyl-SS-AEs as well?…”

“…developed a heterologous system for producing alkylsuccinates in E. coli using a BSS homolog, allowing even more rapid access to substrate scope studies. 34 Although these tools will accelerate the development of XSSs as biocatalysts, a key limitation still existed – in vitro activation and subsequent hydroalkylation using purified enzymes. Whole cell activation for screening of XSS variants is attractive from a high-throughput standpoint.…”

“…XSSs have fascinated and challenged scientists for decades, and numerous studies 5 , 6 , 7 , 21 , 23 , 24 , 25 , 26 , 27 , 28 , 34 have helped to shed light on their mechanism. Methods developments reported here and described above are likely to rapidly accelerate our understanding of XSS enzyme mechanisms, XSS substrate scope, and enable XSS protein engineering efforts.…”

If It Is Hard, It Is Worth Doing: Engineering Radical Enzymes from Anaerobes

“…Mutants of the repair protein IscR have exhibited significantly improved biosynthetic activity for a number of pathways involving radical enzymes, primarily through mitigating the depletion of FeS clusters caused by radical enzyme overexpression. As a result, either incorporation of these mutants or, alternatively, complete deletion of the IscR gene is now becoming a standard technique for in vivo production improvement. − …”

“…There are a number of emerging radical enzyme candidates of potential industrial interest for which the mechanistic data required for rational design is now available. Such enzymes include phenylacetate decarboxylase (PhdB), a glycyl-radical enzyme that is able to produce toluene from renewable resources; the huge range of AdoMet radical enzymes involved in ribosomally synthesized and post-translationally modified peptide (RiPP) biosynthesis pathways, which offer scope for new antimicrobials among other interesting activities; ,, AdoMet enzymes such as the sulfatase AtsB, which can be utilized to create labeled peptides; and many others that may be involved in, for example, environmental detoxification. , …”