Parageobacillus thermoglucosidasius Strain Engineering Using a Theophylline Responsive RiboCas for Controlled Gene Expression.

The relentless increase in atmospheric greenhouse gas concentrations as a consequence of the exploitation of fossil resources compels the adoption of sustainable routes to chemical and fuel manufacture based on biological fermentation processes. The use of thermophilic chassis in such processes is an attractive proposition; however, their effective exploitation will require improved genome editing tools. In the case of the industrially relevant chassis Parageobacillus thermoglucosidasius, CRISPR/Cas9-based gene editing has been demonstrated. The constitutive promoter used, however, accentuates the deleterious nature of Cas9, causing decreased transformation and low editing efficiencies, together with an increased likelihood of off-target effects or alternative mutations. Here, we rectify this issue by controlling the expression of Cas9 through the use of a synthetic riboswitch that is dependent on the nonmetabolized, nontoxic, and cheap inducer, theophylline. We demonstrate that the riboswitches are dose-dependent, allowing for controlled expression of the target gene. Through their use, we were then able to address the deleterious nature of Cas9 and produce an inducible system, RiboCas93. The benefits of RiboCas93 were demonstrated by increased transformation efficiency of the editing vectors, improved efficiency in mutant generation (100%), and a reduction of Cas9 toxicity, as indicated by a reduction in the number of single nucleotide polymorphisms (SNPs) observed. This new system provides a quick and efficient way to produce mutants in P. thermoglucosidasius.

An Updated View on the Cellular Uptake and Mode-of-Action of Clostridioides difficile Toxins.

Research on the human gut pathogen Clostridioides (C.) difficile and its toxins continues to attract much attention as a consequence of the threat to human health posed by hypervirulent strains. Toxin A (TcdA) and Toxin B (TcdB) are the two major virulence determinants of C. difficile. Both are single-chain proteins with a similar multidomain architecture. Certain hypervirulent C. difficile strains also produce a third toxin, namely binary toxin CDT (C. difficile transferase). C. difficile toxins are the causative agents of C. difficile-associated diseases (CDADs), such as antibiotics-associated diarrhea and pseudomembranous colitis. For that reason, considerable efforts have been expended to unravel their molecular mode-of-action and the cellular mechanisms responsible for their uptake. Many of these studies have been conducted in European laboratories. Here, we provide an update on our previous review (Papatheodorou et al. Adv Exp Med Biol, 2018) on important advances in C. difficile toxins research.

A sporulation signature protease is required for assembly of the spore surface layers, germination and host colonization in Clostridioides difficile.

A genomic signature for endosporulation includes a gene coding for a protease, YabG, which in the model organism Bacillus subtilis is involved in assembly of the spore coat. We show that in the human pathogen Clostridioidesm difficile, YabG is critical for the assembly of the coat and exosporium layers of spores. YabG is produced during sporulation under the control of the mother cell-specific regulators σE and σK and associates with the spore surface layers. YabG shows an N-terminal SH3-like domain and a C-terminal domain that resembles single domain response regulators, such as CheY, yet is atypical in that the conserved phosphoryl-acceptor residue is absent. Instead, the CheY-like domain carries residues required for activity, including Cys207 and His161, the homologues of which form a catalytic diad in the B. subtilis protein, and also Asp162. The substitution of any of these residues by Ala, eliminates an auto-proteolytic activity as well as interdomain processing of CspBA, a reaction that releases the CspB protease, required for proper spore germination. An in-frame deletion of yabG or an allele coding for an inactive protein, yabGC207A, both cause misassemby of the coat and exosporium and the formation of spores that are more permeable to lysozyme and impaired in germination and host colonization. Furthermore, we show that YabG is required for the expression of at least two σK-dependent genes, cotA, coding for a coat protein, and cdeM, coding for a key determinant of exosporium assembly. Thus, YabG also impinges upon the genetic program of the mother cell possibly by eliminating a transcriptional repressor. Although this activity has not been described for the B. subtilis protein and most of the YabG substrates vary among sporeformers, the general role of the protease in the assembly of the spore surface is likely to be conserved across evolutionary distance.

Chromosomal engineering of inducible isopropanol- butanol-ethanol production in Clostridium acetobutylicum.

The use of environmentally damaging petrochemical feedstocks can be displaced by fermentation processes based on engineered microbial chassis that recycle biomass-derived carbon into chemicals and fuels. The stable retention of introduced genes, designed to extend product range and/or increase productivity, is essential. Accordingly, we have created multiply marked auxotrophic strains of Clostridium acetobutylicum that provide distinct loci (pyrE, argH, purD, pheA) at which heterologous genes can be rapidly integrated using allele-coupled exchange (ACE). For each locus, ACE-mediated insertion is conveniently selected on the basis of the restoration of prototrophy on minimal media. The Clostridioides difficile gene (tcdR) encoding an orthogonal sigma factor (TcdR) was integrated at the pyrE locus under the control of the lactose-inducible, bgaR::PbgaL promoter to allow the simultaneous control of genes/operons inserted at other disparate loci (purD and pheA) that had been placed under the control of the PtcdB promoter. In control experiments, dose-dependent expression of a catP reporter gene was observed with increasing lactose concentration. At the highest doses tested (10 mM) the level of expression was over 10-fold higher than if catP was placed directly under the control of bgaR::PbgaL and over 2-fold greater than achieved using the strong Pfdx promoter of the Clostridium sporogenes ferredoxin gene. The utility of the system was demonstrated in the production of isopropanol by the C. acetobutylicum strain carrying an integrated copy of tcdR following the insertion of a synthetic acetone operon (ctfA/B, adc) at the purD locus and a gene (sadh) encoding a secondary dehydrogenase at pheA. Lactose induction (10 mM) resulted in the production of 4.4 g/L isopropanol and 19.8 g/L Isopropanol-Butanol-Ethanol mixture.

Endogenous CRISPR/Cas systems for genome engineering in the acetogens Acetobacterium woodii and Clostridium autoethanogenum.

Acetogenic bacteria can play a major role in achieving Net Zero through their ability to convert CO2 into industrially relevant chemicals and fuels. Full exploitation of this potential will be reliant on effective metabolic engineering tools, such as those based on the Streptococcus pyogenes CRISPR/Cas9 system. However, attempts to introduce cas9-containing vectors into Acetobacterium woodii were unsuccessful, most likely as a consequence of Cas9 nuclease toxicity and the presence of a recognition site for an endogenous A. woodii restriction-modification (R-M) system in the cas9 gene. As an alternative, this study aims to facilitate the exploitation of CRISPR/Cas endogenous systems as genome engineering tools. Accordingly, a Python script was developed to automate the prediction of protospacer adjacent motif (PAM) sequences and used to identify PAM candidates of the A. woodii Type I-B CRISPR/Cas system. The identified PAMs and the native leader sequence were characterized in vivo by interference assay and RT-qPCR, respectively. Expression of synthetic CRISPR arrays, consisting of the native leader sequence, direct repeats, and adequate spacer, along with an editing template for homologous recombination, successfully led to the creation of 300 bp and 354 bp in-frame deletions of pyrE and pheA, respectively. To further validate the method, a 3.2 kb deletion of hsdR1 was also generated, as well as the knock-in of the fluorescence-activating and absorption-shifting tag (FAST) reporter gene at the pheA locus. Homology arm length, cell density, and the amount of DNA used for transformation were found to significantly impact editing efficiencies. The devised workflow was subsequently applied to the Type I-B CRISPR/Cas system of Clostridium autoethanogenum, enabling the generation of a 561 bp in-frame deletion of pyrE with 100% editing efficiency. This is the first report of genome engineering of both A. woodii and C. autoethanogenum using their endogenous CRISPR/Cas systems.

Base editing enables duplex point mutagenesis in Clostridium autoethanogenum at the price of numerous off-target mutations.

Base editors are recent multiplex gene editing tools derived from the Cas9 nuclease of Streptomyces pyogenes. They can target and modify a single nucleotide in the genome without inducing double-strand breaks (DSB) of the DNA helix. As such, they hold great potential for the engineering of microbes that lack effective DSB repair pathways such as homologous recombination (HR) or non-homologous end-joining (NHEJ). However, few applications of base editors have been reported in prokaryotes to date, and their advantages and drawbacks have not been systematically reported. Here, we used the base editors Target-AID and Target-AID-NG to introduce nonsense mutations into four different coding sequences of the industrially relevant Gram-positive bacterium Clostridium autoethanogenum. While up to two loci could be edited simultaneously using a variety of multiplexing strategies, most colonies exhibited mixed genotypes and most available protospacers led to undesired mutations within the targeted editing window. Additionally, fifteen off-target mutations were detected by sequencing the genome of the resulting strain, among them seven single-nucleotide polymorphisms (SNP) in or near loci bearing some similarity with the targeted protospacers, one 15 nt duplication, and one 12 kb deletion which removed uracil DNA glycosylase (UDG), a key DNA repair enzyme thought to be an obstacle to base editing mutagenesis. A strategy to process prokaryotic single-guide RNA arrays by exploiting tRNA maturation mechanisms is also illustrated.

Pathogenicity and virulence of Clostridium botulinum.

Clostridium botulinum, a polyphyletic Gram-positive taxon of bacteria, is classified purely by their ability to produce botulinum neurotoxin (BoNT). BoNT is the primary virulence factor and the causative agent of botulism. A potentially fatal disease, botulism is classically characterized by a symmetrical descending flaccid paralysis, which is left untreated can lead to respiratory failure and death. Botulism cases are classified into three main forms dependent on the nature of intoxication; foodborne, wound and infant. The BoNT, regarded as the most potent biological substance known, is a zinc metalloprotease that specifically cleaves SNARE proteins at neuromuscular junctions, preventing exocytosis of neurotransmitters, leading to muscle paralysis. The BoNT is now used to treat numerous medical conditions caused by overactive or spastic muscles and is extensively used in the cosmetic industry due to its high specificity and the exceedingly small doses needed to exert long-lasting pharmacological effects. Additionally, the ability to form endospores is critical to the pathogenicity of the bacteria. Disease transmission is often facilitated via the metabolically dormant spores that are highly resistant to environment stresses, allowing persistence in the environment in unfavourable conditions. Infant and wound botulism infections are initiated upon germination of the spores into neurotoxin producing vegetative cells, whereas foodborne botulism is attributed to ingestion of preformed BoNT. C. botulinum is a saprophytic bacterium, thought to have evolved its potent neurotoxin to establish a source of nutrients by killing its host.

Metabolic engineering for the production of acetoin and 2,3-butanediol at elevated temperature in Parageobacillus thermoglucosidasius NCIMB 11955.

The current climate crisis has emphasised the need to achieve global net-zero by 2050, with countries being urged to set considerable emission reduction targets by 2030. Exploitation of a fermentative process that uses a thermophilic chassis can represent a way to manufacture chemicals and fuels through more environmentally friendly routes with a net reduction in greenhouse gas emissions. In this study, the industrially relevant thermophile Parageobacillus thermoglucosidasius NCIMB 11955 was engineered to produce 3-hydroxybutanone (acetoin) and 2,3-butanediol (2,3-BDO), organic compounds with commercial applications. Using heterologous acetolactate synthase (ALS) and acetolactate decarboxylase (ALD) enzymes, a functional 2,3-BDO biosynthetic pathway was constructed. The formation of by-products was minimized by the deletion of competing pathways surrounding the pyruvate node. Redox imbalance was addressed through autonomous overexpression of the butanediol dehydrogenase and by investigating appropriate aeration levels. Through this, we were able to produce 2,3-BDO as the predominant fermentation metabolite, with up to 6.6 g/L 2,3-BDO (0.33 g/g glucose) representing 66% of the theoretical maximum at 50°C. In addition, the identification and subsequent deletion of a previously unreported thermophilic acetoin degradation gene (acoB1) resulted in enhanced acetoin production under aerobic conditions, producing 7.6 g/L (0.38 g/g glucose) representing 78% of the theoretical maximum. Furthermore, through the generation of a ΔacoB1 mutant and by testing the effect of glucose concentration on 2,3-BDO production, we were able to produce 15.6 g/L of 2,3-BDO in media supplemented with 5% glucose, the highest titre of 2,3-BDO produced in Parageobacillus and Geobacillus species to date.

Clostridioides difficile Binary Toxin Binding Component Increases Virulence in a Hamster Model.

Background: Clostridioides difficile is the leading cause of hospital-acquired gastrointestinal infection, in part due to the existence of binary toxin (CDT)-expressing hypervirulent strains. Although the effects of the CDT holotoxin on disease pathogenesis have been previously studied, we sought to investigate the role of the individual components of CDT during in vivo infection. Methods: To determine the contribution of the separate components of CDT during infection, we developed strains of C difficile expressing either CDTa or CDTb individually. We then infected both mice and hamsters with these novel mutant strains and monitored them for development of severe illness. Results: Although expression of CDTb without CDTa did not induce significant disease in a mouse model of C difficile infection, we found that complementation of a CDT-deficient C difficile strain with CDTb alone restored virulence in a hamster model of C difficile infection. Conclusions: Overall, this study demonstrates that the binding component of C difficile binary toxin, CDTb, contributes to virulence in a hamster model of infection.

In Vivo Genome Editing in Type I and II Methanotrophs Using a CRISPR/Cas9 System.

Methanotrophic bacteria are Gram-negative, aerobic organisms that use methane as their sole source of carbon and energy. In this study, we constructed and exemplified a CRISPR/Cas9 genome editing system and used it to successfully make gene deletions and insertions in the type I methanotroph Methylococcus capsulatus Bath and the type II methanotroph Methylocystis parvus OBBP. High frequencies of gene deletions and insertions were achieved in combination with homology-directed repair. In M. parvus OBBP, we also investigated the impact of several parameters on the CRISPR/Cas9 genome editing, where the ligD gene was targeted with various PAM sequences and guide RNA spacer sequences, homology arms of variable length, differences in the duration of mating during conjugation, and exploiting promoters of different strengths to control the expression of cas9 and sgRNA. Although not the first attempt to develop a CRISPR/Cas system in methanotrophs, this work demonstrated for the first time an efficient CRISPR/Cas9 system generating scarless clean gene deletions and insertions in methanotroph genomes.

Metabolic engineering of Cupriavidus necator H16 for heterotrophic and autotrophic production of 3-hydroxypropionic acid.

3-Hydroxypropionate (3-HP) is a versatile compound for chemical synthesis and a potential building block for biodegradable polymers. Cupriavidus necator H16, a facultative chemolithoautotroph, is an attractive production chassis and has been extensively studied as a model organism for biopolymer production. Here, we engineered C. necator H16 for 3-HP biosynthesis from its central metabolism. Wild type C. necator H16 can use 3-HP as a carbon source, a highly undesirable trait for a 3-HP production chassis. However, deletion of its three (methyl-)malonate semialdehyde dehydrogenases (mmsA1, mmsA2 and mmsA3) resulted in a strain that cannot grow on 3-HP as the sole carbon source, and this strain was selected as our production host. A stepwise approach was used to construct pathways for 3-HP production via ß-alanine. Two additional gene deletion targets were identified during the pathway construction process. Deletion of the 3-hydroxypropionate dehydrogenase, encoded by hpdH, prevented the re-consumption of the 3-HP produced by our engineered strains, while deletion of gdhA1, annotated as a glutamate dehydrogenase, prevented the utilization of aspartate as a carbon source, one of the key pathway intermediates. The final strain carrying these deletions was able to produce up to 8 mM 3-HP heterotrophically. Furthermore, an engineered strain was able to produce 0.5 mM 3-HP under autotrophic conditions, using CO2 as sole carbon source. These results form the basis for establishing C. necator H16 as an efficient platform for the production of 3-HP and 3-HP-containing polymers.

pMTL60000: A modular plasmid vector series for Parageobacillus thermoglucosidasius strain engineering.

Parageobacillus thermoglucosidasius is a promising chassis for producing chemicals and fuels. Here we designed, built and tested the pMTL60000 modular plasmids containing standardised Gram-positive and Gram-negative replicons, selectable markers and application-specific modules. The pMTL60000 modular plasmids were characterised with regard to transformation efficiency, segregational stability, copy number and compatibility.

Genome Sequence of Eubacterium limosum B2 and Evolution for Growth on a Mineral Medium with Methanol and CO2 as Sole Carbon Sources.

Eubacterium limosum is an acetogen that can produce butyrate along with acetate as the main fermentation end-product from methanol, a promising C1 feedstock. Although physiological characterization of E. limosum B2 during methylotrophy was previously performed, the strain was cultured in a semi-defined medium, limiting the scope for further metabolic insights. Here, we sequenced the complete genome of the native strain and performed adaptive laboratory evolution to sustain growth on methanol mineral medium. The evolved population significantly improved its maximal growth rate by 3.45-fold. Furthermore, three clones from the evolved population were isolated on methanol mineral medium without cysteine by the addition of sodium thiosulfate. To identify mutations related to growth improvement, the whole genomes of wild-type E. limosum B2, the 10th, 25th, 50th, and 75th generations, and the three clones were sequenced. We explored the total proteomes of the native and the best evolved clone (n°2) and noticed significant differences in proteins involved in gluconeogenesis, anaplerotic reactions, and sulphate metabolism. Furthermore, a homologous recombination was found in subunit S of the type I restriction-modification system between both strains, changing the structure of the subunit, its sequence recognition and the methylome of the evolved clone. Taken together, the genomic, proteomic and methylomic data suggest a possible epigenetic mechanism of metabolic regulation.

Inducible CRISPR/Cas9 Allows for Multiplexed and Rapidly Segregated Single-Target Genome Editing in Synechocystis Sp. PCC 6803.

Establishing various synthetic biology tools is crucial for the development of cyanobacteria for biotechnology use, especially tools that allow for precise and markerless genome editing in a time-efficient manner. Here, we describe a riboswitch-inducible CRISPR/Cas9 system, contained on a single replicative vector, for the model cyanobacterium Synechocystis sp. PCC 6803. A theophylline-responsive riboswitch allowed tight control of Cas9 expression, which enabled reliable transformation of the CRISPR/Cas9 vector intoSynechocystis. Induction of the CRISPR/Cas9 mediated various types of genomic edits, specifically deletions and insertions of varying size. The editing efficiency varied depending on the target and intended edit; smaller edits performed better, reaching, e.g., 100% for insertion of a FLAG-tag onto rbcL. Importantly, the single-vector CRISPR/Cas9 system mediated multiplexed editing of up to three targets in parallel inSynechocystis. All single-target and several double-target mutants were also fully segregated after the first round of induction. Lastly, a vector curing system based on the nickel-inducible expression of the toxic mazF (from Escherichia coli) was added to the CRISPR/Cas9 vector. This inducible system allowed for curing of the vector in 25-75% of screened colonies, enabling edited mutants to become markerless.

A clean in-frame knockout system for gene deletion in Acetobacterium woodii.

Acetogenic bacteria produce acetate following the fixation of CO2 via the Wood-Ljungdahl pathway. As such, they represent excellent process organisms for the production of novel chemicals and fuels from this waste greenhouse gas. Acetobacterium woodii is the model acetogen and numerous studies have been conducted investigating its biochemistry, gas consumption and use as a production chassis. However, there are a dearth of available tools for A. woodii gene modification which limits the research options available for genetic studies. Here, the previously proposed Clostridia Roadmap is implemented in A. woodii leading to the derivation of a knockout system for the generation of clean, in-frame deletions. The replicon of the Gram-positive plasmid pCD6 that originated in Clostridioides difficile was identified as being replication-defective in A. woodii, a property that was exploited to construct a pseudo-suicide knockout plasmid which was used to generate an auxotrophic, pyrE mutant. This allowed the subsequent use of a heterologous pyrE gene (from Clostridium acetobutylicum) as a counter selection marker and the deletion of a number of genes by allelic exchange. Specific mutants generated were affected in growth on glucose, fructose and ethanol as a consequence of deletion of fruA, pstG and adhE, respectively.

A genome-scale metabolic model of Cupriavidus necator H16 integrated with TraDIS and transcriptomic data reveals metabolic insights for biotechnological applications.

Exploiting biological processes to recycle renewable carbon into high value platform chemicals provides a sustainable and greener alternative to current reliance on petrochemicals. In this regard Cupriavidus necator H16 represents a particularly promising microbial chassis due to its ability to grow on a wide range of low-cost feedstocks, including the waste gas carbon dioxide, whilst also naturally producing large quantities of polyhydroxybutyrate (PHB) during nutrient-limited conditions. Understanding the complex metabolic behaviour of this bacterium is a prerequisite for the design of successful engineering strategies for optimising product yields. We present a genome-scale metabolic model (GSM) of C. necator H16 (denoted iCN1361), which is directly constructed from the BioCyc database to improve the readability and reusability of the model. After the initial automated construction, we have performed extensive curation and both theoretical and experimental validation. By carrying out a genome-wide essentiality screening using a Transposon-directed Insertion site Sequencing (TraDIS) approach, we showed that the model could predict gene knockout phenotypes with a high level of accuracy. Importantly, we indicate how experimental and computational predictions can be used to improve model structure and, thus, model accuracy as well as to evaluate potential false positives identified in the experiments. Finally, by integrating transcriptomics data with iCN1361 we create a condition-specific model, which, importantly, better reflects PHB production in C. necator H16. Observed changes in the omics data and in-silico-estimated alterations in fluxes were then used to predict the regulatory control of key cellular processes. The results presented demonstrate that iCN1361 is a valuable tool for unravelling the system-level metabolic behaviour of C. necator H16 and can provide useful insights for designing metabolic engineering strategies.

Design, Analysis, and Implementation of a Novel Biochemical Pathway for Ethylene Glycol Production in Clostridium autoethanogenum.

The platform chemical ethylene glycol (EG) is used to manufacture various commodity chemicals of industrial importance, but largely remains synthesized from fossil fuels. Although several novel metabolic pathways have been reported for its bioproduction in model organisms, none has been reported for gas-fermenting, non-model acetogenic chassis organisms. Here, we describe a novel, synthetic biochemical pathway to convert acetate into EG in the industrially important gas-fermenting acetogen,Clostridium autoethanogenum. We not only developed a computational workflow to design and analyze hundreds of novel biochemical pathways for EG production but also demonstrated a successful pathway construction in the chosen host. The EG production was achieved using a two-plasmid system to bypass unfeasible expression levels and potential toxic enzymatic interactions. Although only a yield of 0.029 g EG/g fructose was achieved and therefore requiring further strain engineering efforts to optimize the designed strain, this work demonstrates an important proof-of-concept approach to computationally design and experimentally implement fully synthetic metabolic pathways in a metabolically highly specific, non-model host organism.

Required Gene Set for Autotrophic Growth of Clostridium autoethanogenum.

The majority of the genes present in bacterial genomes remain poorly characterized, with up to one-third of those that are protein encoding having no definitive function. Transposon insertion sequencing represents a high-throughput technique that can help rectify this deficiency. The technology, however, can only be realistically applied to those species in which high rates of DNA transfer can be achieved. Here, we have developed a number of approaches that overcome this barrier in the autotrophic species Clostridium autoethanogenum by using a mariner-based transposon system. The inherent instability of such systems in the Escherichia coli conjugation donor due to transposition events was counteracted through the incorporation of a conditionally lethal codA marker on the plasmid backbone. Relatively low frequencies of transformation of the plasmid into C. autoethanogenum were circumvented through the use of a plasmid that is conditional for replication coupled with the routine implementation of an Illumina library preparation protocol that eliminates plasmid-based reads. A transposon library was then used to determine the essential genes needed for growth using carbon monoxide as the sole carbon and energy source. IMPORTANCE Although microbial genome sequences are relatively easily determined, assigning gene function remains a bottleneck. Consequently, relatively few genes are well characterized, leaving the function of many as either hypothetical or entirely unknown. High-throughput transposon sequencing can help remedy this deficiency, but is generally only applicable to microbes with efficient DNA transfer procedures. These exclude many microorganisms of importance to humankind either as agents of disease or as industrial process organisms. Here, we developed approaches to facilitate transposon insertion sequencing in the acetogen Clostridium autoethanogenum, a chassis being exploited to convert single-carbon waste gases CO and CO2 into chemicals and fuels at an industrial scale. This allowed the determination of gene essentiality under heterotrophic and autotrophic growth, providing insights into the utilization of CO as a sole carbon and energy source. The strategies implemented are translatable and will allow others to apply transposon insertion sequencing to other microbes where DNA transfer has until now represented a barrier to progress.

Biosensor-informed engineering of Cupriavidus necator H16 for autotrophic D-mannitol production.

Cupriavidus necator H16 is one of the most researched carbon dioxide (CO2)-fixing bacteria. It can store carbon in form of the polymer polyhydroxybutyrate and generate energy by aerobic hydrogen oxidation under lithoautotrophic conditions, making C. necator an ideal chassis for the biological production of value-added compounds from waste gases. Despite its immense potential, however, the experimental evidence of C. necator utilisation for autotrophic biosynthesis of chemicals is limited. Here, we genetically engineered C. necator for the high-level de novo biosynthesis of the industrially relevant sugar alcohol mannitol directly from Calvin-Benson-Bassham (CBB) cycle intermediates. To identify optimal mannitol production conditions in C. necator, a mannitol-responsive biosensor was applied for screening of mono- and bifunctional mannitol 1-phosphate dehydrogenases (MtlDs) and mannitol 1-phosphate phosphatases (M1Ps). We found that MtlD/M1P from brown alga Ectocarpus siliculosus performed overall the best under heterotrophic growth conditions and was selected to be chromosomally integrated. Consequently, autotrophic fermentation of recombinant C. necator yielded up to 3.9 g/L mannitol, representing a substantial improvement over mannitol biosynthesis using recombinant cyanobacteria. Importantly, we demonstrate that at the onset of stationary growth phase nearly 100% of carbon can be directed from the CBB cycle into mannitol through the glyceraldehyde 3-phosphate and fructose 6-phosphate intermediates. This study highlights for the first time the potential of C. necator to generate sugar alcohols from CO2 utilising precursors derived from the CBB cycle.