Part by Part: Synthetic Biology Parts Used in Solventogenic Clostridia

Abstract

The solventogenic Clostridia are of interest to the chemical industry because of their natural ability to produce chemicals such as butanol, acetone and ethanol from diverse feedstocks. Their use as whole cell factories presents multiple metabolic engineering targets that could lead to improved sustainability and profitability of Clostridium industrial processes. However, engineering efforts have been held back by the scarcity of genetic and synthetic biology tools. Over the past decade, genetic tools to enable transformation and chromosomal modifications have been developed, but the lack of a broad palette of synthetic biology parts remains one of the last obstacles to the rapid engineered improvement of these species for bioproduction. We have systematically reviewed existing parts that have been used in the modification of solventogenic Clostridia, revealing a narrow range of empirically chosen and nonengineered parts that are in current use. The analysis uncovers elements, such as promoters, transcriptional terminators and ribosome binding sites where increased fundamental knowledge is needed for their reliable use in different applications. Together, the review provides the most comprehensive list of parts used and also presents areas where an improved toolbox is needed for full exploitation of these industrially important bacteria.

Robust microorganisms for biofuel and chemical production from municipal solid waste

“…Extracting tannins from the OMSW prior to hydrolysis could alleviate inhibition in C. acetobutylicum [15], however, on industrial scales this would require additional processing steps that may be uneconomical. Another option would be to engineer phenol tolerance using the ever increasing repertoire or genetic and synthetic biology tools available for Clostridia [82].…”

Stringency of Synthetic Promoter Sequences in Clostridium Revealed and Circumvented by Tuning Promoter Library Mutation Rates

“…However, metabolic engineering using such elements has not been well established in Clostridium . Instead, protein expression has relied upon natural promoter sequences such as the C. acetobutylicum thiolase gene promoter, , C. sporogenes ferredoxin gene promoter, phosphotransbutylase gene promoter , or acetoacetate decarboxylase gene promoter, ,, which are discussed in a recent review . However, natural promoters offer only a limited range of specific strengths and are typically subject to native regulation and variation under changing conditions .…”

“…Instead, protein expression has relied upon natural promoter sequences such as the C. acetobutylicum thiolase gene promoter, 14,15 C. sporogenes ferredoxin gene promoter, 4 phosphotransbutylase gene promoter 16,17 or acetoacetate decarboxylase gene promoter, 15,16,18 which are discussed in a recent review. 19 However, natural promoters offer only a limited range of specific strengths and are typically subject to native regulation and variation under changing conditions. 20 In other organisms, the limitations of natural promoters have been avoided by generating synthetic promoter libraries using the approach first described by Jensen and Hammer.…”

A Gold Standard, CRISPR/Cas9-Based Complementation Strategy Reliant on 24 Nucleotide Bookmark Sequences

“…The genus Clostridium encompasses many species of medical and industrial interest, for instance, Clostridium difficile [1], Clostridium botulinum [2], Clostridium acetobutylicum [3] and Clostridium autoethanogenum [4]. Accordingly, substantial efforts have been expended in recent years to develop convenient and standardized genetic toolboxes to enable the systematic study and engineering of clostridia [5][6][7][8]. A fundamental requirement has been the ability to establish gene function through precise gene knock-out (KO) and the subsequent phenotypic comparison of the mutant KO and progenitor strain, typically, the Wild-Type (WT).…”