Dr Liz Jenkinson

Abstract:

The development and advent of mutagenesis tools for solventogenic clostridial species in recent years has allowed for the increased refinement of industrially relevant strains. In this study we have utilised CLEAVE™, a CRISPR/Cas genome editing system developed by Green Biologics Ltd., to engineer a strain of Clostridium saccharoperbutylacetonicum N1-4(HMT) with potentially useful solvents titres and energy metabolism. As one of two enzymes responsible for the conversion of glyceraldehyde-3-phosphate (GAP) to 3-phosphoglyceric acid in glycolysis, it was hypothesised that deletion of gapN would increase ATP and NADH production that could in turn improve solvent production. Herein, whole genome sequencing has been used to evaluate CLEAVE™ and the successful knockout of gapN, demonstrating a clean knockout with no other detectable variations from the wild type sequence. Elevated solvent levels were detected during the first 24 h of batch fermentation, indicating an earlier shift to solventogenesis. A 2.4-fold increase in ATP concentration was observed, and quantitation of NAD(P)H derivatives revealed a more reducing cytoplasm for the gapN strain. These findings expand our understanding of clostridium carbon metabolism and report a new approach to optimising biofuel production.

Abstract:

Butyrate is produced by chemical synthesis based on crude oil, produced by microbial fermentation, or extracted from animal fats (M. Dwidar, J.-Y. Park, R. J. Mitchell, and B.-I. Sang, The Scientific World Journal, 2012:471417, 2012, https://doi.org/10.1100/2012/471417). Butyrate production by anaerobic bacteria is highly favorable since waste or sustainable resources can be used as the substrates. For this purpose, the native hyper-butanol producer Clostridium saccharoperbutylacetonicum N1-4(HMT) was used as a chassis strain due to its broad substrate spectrum. BLASTp analysis of the predicted proteome of C. saccharoperbutylacetonicum N1-4(HMT) resulted in the identification of gene products potentially involved in acetone-butanol-ethanol (ABE) fermentation. Their participation in ABE fermentation was either confirmed or disproven by the parallel production of acids or solvents and the respective transcript levels obtained by transcriptome analysis of this strain. The genes encoding phosphotransacetylase (pta) and butyraldehyde dehydrogenase (bld) were deleted to reduce acetate and alcohol formation. The genes located in the butyryl-CoA synthesis (bcs) operon encoding crotonase, butyryl-CoA dehydrogenase with electron-transferring protein subunits α and β, and 3-hydroxybutyryl-CoA dehydrogenase were overexpressed to channel the flux further towards butyrate formation. Thereby, the native hyper-butanol producer C. saccharoperbutylacetonicum N1-4(HMT) was converted into the hyper-butyrate producer C. saccharoperbutylacetonicum ΔbldΔpta [pMTL83151_BCS_P_bgaL]. The transcription pattern following deletion and overexpression was characterized by a second transcriptomic study, revealing partial compensation for the deletion. Furthermore, this strain was characterized in pH-controlled fermentations with either glucose or Excello, a substrate yielded from spruce biomass. Butyrate was the main product, with maximum butyrate concentrations of 11.7 g·L^-1 and 14.3 g·L^-1, respectively. Minimal amounts of by-products were detected. IMPORTANCE Platform chemicals such as butyrate are usually produced chemically from crude oil, resulting in the carry-over of harmful compounds. The selective production of butyrate using sustainable resources or waste without harmful by-products can be achieved by bacteria such as clostridia. The hyper-butanol producer Clostridium saccharoperbutylacetonicum N1-4(HMT) was converted into a hyper-butyrate producer. Butyrate production with very small amounts of by-products was established with glucose and the sustainable lignocellulosic sugar substrate Excello extracted from spruce biomass by the biorefinery Borregaard (Sarpsborg, Norway).

Abstract:

The solventogenic clostridia have long been known for their ability to convert sugars from complex feedstocks into commercially important solvents. Although the acetone-butanol-ethanol process fell out of favour decades ago, renewed interest in sustainability and 'green' chemistry has re-established our appetite for reviving technologies such as these, albeit with 21st century improvements. As CRISPR-Cas genome editing tools are being developed and applied to the solventogenic clostridia, their industrial potential is growing. Through integration of new pathways, the beneficial traits and historical track record of clostridial fermentation can be exploited to generate a much wider range of industrially relevant products. Here we show the application of genome editing using the endogenous CRISPR-Cas mechanism of Clostridium saccharoperbutylacetonicum N1-4(HMT), to generate a deletion, SNP and to integrate new DNA into the genome. These technological advancements pave the way for application of clostridial species to the production of an array of products.

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.