Furan-2,5-dicarboxylic acid (FDCA) is a biobased platform chemical for the production of polymers. In the past few years, numerous multistep chemical routes have been reported on the synthesis of FDCA by oxidation of 5-hydroxymethylfurfural (HMF). Recently we identified an FAD-dependent enzyme which is active towards HMF and related compounds. This oxidase has the remarkable capability of oxidizing [5-(hydroxymethyl)furan-2-yl]methanol to FDCA, a reaction involving four consecutive oxidations. The oxidase can produce FDCA from HMF with high yield at ambient temperature and pressure. Examination of the underlying mechanism shows that the oxidase acts on alcohol groups only and depends on the hydration of aldehydes for the oxidation reaction required to form FDCA.

Members of the genus Clostridium represent a diverse assemblage of species exhibiting both medical and industrial importance. Deriving both a greater understanding of their biology, while at the same time enhancing their exploitable properties, requires effective genome editing tools. Here, we demonstrate the first implementation in the genus of theophylline-dependent, synthetic riboswitches exhibiting a full set of dynamic ranges, also suitable for applications where tight control of gene expression is required. Their utility was highlighted by generating a novel riboswitch-based editing toolRiboCasthat overcomes the main obstacles associated with CRISPR/Cas9 systems, including low transformation efficiencies and excessive Cas9 toxicity. The universal nature of the tool was established by obtaining chromosomal modifications in C. pasteurianum, C. difficile, and C. sporogenes, as well as by carrying out the first reported example of CRISPR-targeted gene disruption in C. botulinum. The high efficiency (100% mutant generation) and ease of application of RiboCas make it suitable for use in a diverse range of microorganisms.

Increasing protein expression levels is a key step in the commercial production of enzymes. Predicting promoter activity and translation initiation efficiency based solely on consensus sequences have so far met with mixed results. Here, we addressed this challenge using a "brute-force" approach by designing and synthesizing a large combinatorial library comprising ∼12 000 unique synthetic expression modules (SEMs) for Bacillus subtilis. Using GFP fluorescence as a reporter of gene expression, we obtained a dynamic expression range that spanned 5 orders of magnitude, as well as a maximal 13-fold increase in expression compared with that of the already strong veg expression module. Analyses of the synthetic modules indicated that sequences at the 5'-end of the mRNA were the most important contributing factor to the differences in expression levels, presumably by preventing formation of strong secondary mRNA structures that affect translation initiation. When the gfp coding region was replaced by the coding region of the xynA gene, encoding the industrially relevant B. subtilis xylanase enzyme, only a 3-fold improvement in xylanase production was observed. Moreover, the correlation between GFP and xylanase expression levels was weak. This suggests that the differences in expression levels between the gfp and xynA constructs were due to differences in 5'-end mRNA folding and consequential differences in the rates of translation initiation. Our data show that the use of large libraries of SEMs, in combination with high-throughput technologies, is a powerful approach to improve the production of a specific protein, but that the outcome cannot necessarily be extrapolated to other proteins.