Strain engineering and bioprocessing strategies for biobased production of porphobilinogen in Escherichia coli.

Strain engineering and bioprocessing strategies were applied for biobased production of porphobilinogen (PBG) using Escherichia coli as the cell factory. The non-native Shemin/C4 pathway was first implemented by heterologous expression of hemA from Rhodopseudomonas spheroids to supply carbon flux from the natural tricarboxylic acid (TCA) pathways for PBG biosynthesis via succinyl-CoA. Metabolic strategies were then applied for carbon flux direction from the TCA pathways to the C4 pathway. To promote PBG stability and accumulation, Clustered Regularly Interspersed Short Palindromic Repeats interference (CRISPRi) was applied to repress hemC expression and, therefore, reduce carbon flowthrough toward porphyrin biosynthesis with minimal impact to cell physiology. To further enhance PBG biosynthesis and accumulation under the hemC-repressed genetic background, we further heterologously expressed native E. coli hemB. Using these engineered E. coli strains for bioreactor cultivation based on ~ 30 g L-1 glycerol, we achieved high PBG titers up to 209 mg L-1, representing 1.73% of the theoretical PBG yield, with improved PBG stability and accumulation. Potential biochemical, genetic, and metabolic factors limiting PBG production were systematically identified for characterization. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s40643-021-00482-3.

Bio-based production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) with modulated monomeric fraction in Escherichia coli.

In this study, we applied metabolic engineering and bioprocessing strategies to enhance heterologous production of an important biodegradable copolymer, i.e., poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), with a modulated 3-hydroxyvalerate (3-HV) monomeric fraction from structurally unrelated carbon of glycerol in engineered Escherichia coli under different oxygenic conditions. We used our previously derived propanologenic (i.e., 1-propanol-producing) E. coli strain with an activated genomic Sleeping beauty mutase (Sbm) operon as a host for heterologous expression of the phaCAB operon. The 3-HV monomeric fraction was modulated by regulating dissimilated carbon flux channeling from the tricarboxylic acid (TCA) cycle into the Sbm pathway for biosynthesis of propionyl-CoA, which is a key precursor to (R)-3-hydroxyvaleryl-CoA (3-HV-CoA) monomer. The carbon flux channeling was regulated either by manipulating a selection of genes involved in the TCA cycle or varying oxygenic condition of the bacterial culture. With these consolidated strategies being implemented, we successfully achieved high-level PHBV biosynthesis with a wide range of 3-HV monomeric fraction from ~ 4 to 50 mol%, potentially enabling the fine-tuning of PHBV mechanical properties at the biosynthesis stage. We envision that similar strategies can be applied to enhance bio-based production of chemicals derived from succinyl-CoA. KEY POINTS: • TCA cycle engineering was applied to enhance 3-HV monomeric fraction in E. coli. • Effects of oxygenic conditions on 3-HV incorporation into PHBV in E. coli were investigated. • Bacterial cultivation for high-level PHBV production in engineered E. coli was performed.

Strain engineering for high-level 5-aminolevulinic acid production in Escherichia coli.

Herein, we report the development of a microbial bioprocess for high-level production of 5-aminolevulinic acid (5-ALA), a valuable non-proteinogenic amino acid with multiple applications in medical, agricultural, and food industries, using Escherichia coli as a cell factory. We first implemented the Shemin (i.e., C4) pathway for heterologous 5-ALA biosynthesis in E. coli. To reduce, but not to abolish, the carbon flux toward essential tetrapyrrole/porphyrin biosynthesis, we applied clustered regularly interspersed short palindromic repeats interference (CRISPRi) to repress hemB expression, leading to extracellular 5-ALA accumulation. We then applied metabolic engineering strategies to direct more dissimilated carbon flux toward the key precursor of succinyl-CoA for enhanced 5-ALA biosynthesis. Using these engineered E. coli strains for bioreactor cultivation, we successfully demonstrated high-level 5-ALA biosynthesis from glycerol (~30 g L-1 ) under both microaerobic and aerobic conditions, achieving up to 5.95 g L-1 (36.9% of the theoretical maximum yield) and 6.93 g L-1 (50.9% of the theoretical maximum yield) 5-ALA, respectively. This study represents one of the most effective bio-based production of 5-ALA from a structurally unrelated carbon to date, highlighting the importance of integrated strain engineering and bioprocessing strategies to enhance bio-based production.

Integrated strain engineering and bioprocessing strategies for high-level bio-based production of 3-hydroxyvalerate in Escherichia coli.

As petro-based production generates numerous environmental impacts and their associated technological concerns, bio-based production has been well recognized these days as a modern alternative to manufacture chemical products in a more renewable, environmentally friendly, and sustainable manner. Herein, we report the development of a microbial bioprocess for high-level and potentially economical production of 3-hydroxyvalerate (3-HV), a valuable special chemical with multiple applications in chemical, biopolymer, and pharmaceutical industries, from glycerol, which can be cheaply and renewably refined as a byproduct from biodiesel production. We used our recently derived 3-HV-producing Escherichia coli strains for bioreactor characterization under various culture conditions. In the parental strain, 3-HV biosynthesis was limited by the intracellular availability of propionyl-CoA, whose formation was favored by anaerobic conditions, which often compromised cell growth. With appropriate strain engineering, we demonstrated that 3-HV can be effectively produced under both microaerobic (close to anaerobic) and aerobic conditions, which determine the direction of dissimilated carbon flux toward the succinate node in the tricarboxylic acid (TCA) cycle. We first used the ∆sdhA single mutant strain, in which the dissimilated carbon flux was primarily directed to the Sleeping beauty mutase (Sbm) pathway (via the reductive TCA branch, with enhanced cell growth under microaerobic conditions, achieving 3.08 g L-1 3-HV in a fed-batch culture. In addition, we used the ∆sdhA-∆iclR double mutant strain, in which the dissimilated carbon flux was directed from the TCA cycle to the Sbm pathway via the deregulated glyoxylate shunt, for cultivation under rather aerobic conditions. In addition to demonstrating effective cell growth, this strain has shown impressive 3-HV biosynthesis (up to 10.6 g L-1), equivalent to an overall yield of 18.8% based on consumed glycerol, in aerobic fed-batch culture. This study not only represents one of the most effective bio-based production of 3-HV from structurally unrelated carbons to date, but also highlights the importance of integrated strain engineering and bioprocessing strategies to enhance bio-based production.Key points• TCA cycle engineering was applied to enhance 3-HV biosynthesis in E. coli. • Effects of oxygenic conditions on 3-HV in E. coli biosynthesis were investigated. • Bioreactor characterization of 3-HV biosynthesis in E. coli was performed.

High-level heterologous production of propionate in engineered Escherichia coli.

A propanologenic (i.e., 1-propanol-producing) bacterium Escherichia coli strain was previously derived by activating the genomic sleeping beauty mutase (Sbm) operon. The activated Sbm pathway branches out of the tricarboxylic acid (TCA) cycle at the succinyl-CoA node to form propionyl-CoA and its derived metabolites of 1-propanol and propionate. In this study, we targeted several TCA cycle genes encoding enzymes near the succinyl-CoA node for genetic manipulation to identify the individual contribution of the carbon flux into the Sbm pathway from the three TCA metabolic routes, that is, oxidative TCA cycle, reductive TCA branch, and glyoxylate shunt. For the control strain CPC-Sbm, in which propionate biosynthesis occurred under relatively anaerobic conditions, the carbon flux into the Sbm pathway was primarily derived from the reductive TCA branch, and both succinate availability and the SucCD-mediated interconversion of succinate/succinyl-CoA were critical for such carbon flux redirection. Although the oxidative TCA cycle normally had a minimal contribution to the carbon flux redirection, the glyoxylate shunt could be an alternative and effective carbon flux contributor under aerobic conditions. With mechanistic understanding of such carbon flux redirection, metabolic strategies based on blocking the oxidative TCA cycle (via ∆sdhA mutation) and deregulating the glyoxylate shunt (via ∆iclR mutation) were developed to enhance the carbon flux redirection and therefore propionate biosynthesis, achieving a high propionate titer of 30.9 g/L with an overall propionate yield of 49.7% upon fed-batch cultivation of the double mutant strain CPC-Sbm∆sdhA∆iclR under aerobic conditions. The results also suggest that the Sbm pathway could be metabolically active under both aerobic and anaerobic conditions.

Heterologous production of 3-hydroxyvalerate in engineered Escherichia coli.

3-Hydroxyacids are a group of valuable fine chemicals with numerous applications, and 3-hydroxybutyrate (3-HB) represents the most common species with acetyl-CoA as a precursor. Due to the lack of propionyl-CoA in most, if not all, microorganisms, bio-based production of 3-hydroxyvalerate (3-HV), a longer-chain 3-hydroxyacid member with both acetyl-CoA and propionyl-CoA as two precursors, is often hindered by high costs associated with the supplementation of related carbon sources, such as propionate or valerate. Here, we report the derivation of engineered Escherichia coli strains for the production of 3-HV from unrelated cheap carbon sources, in particular glucose and glycerol. Activation of the sleeping beauty mutase (Sbm) pathway in E. coli enabled the intracellular formation of non-native propionyl-CoA. A selection of enzymes involved in 3-HV biosynthetic pathway from various microorganisms were explored for investigating their effects on 3-HV biosynthesis in E. coli. Glycerol outperformed glucose as the carbon source, and glycerol dissimilation for 3-HV biosynthesis was primarily mediated through the aerobic GlpK-GlpD route. To further enhance 3-HV production, we developed metabolic engineering strategies to redirect more dissimilated carbon flux from the tricarboxylic acid (TCA) cycle to the Sbm pathway, resulting in an enlarged intracellular pool of propionyl-CoA. Both the presence of succinate/succinyl-CoA and their interconversion step in the TCA cycle were identified to critically limit the carbon flux redirection into the Sbm pathway and, therefore, 3-HV biosynthesis. A selection of E. coli host TCA genes encoding enzymes near the succinate node were targeted for manipulation to evaluate the contribution of the three TCA routes (i.e. oxidative TCA cycle, reductive TCA branch, and glyoxylate shunt) to the redirected carbon flux into the Sbm pathway. Finally, the carbon flux redirection into the Sbm pathway was enhanced by simultaneously deregulating glyoxylate shunt and blocking the oxidative TCA cycle, significantly improving 3-HV biosynthesis. With the implementation of these biotechnological and bioprocessing strategies, our engineered E. coli strains can effectively produce 3-HV up to 3.71 g l-1 with a yield of 24.1% based on the consumed glycerol in shake-flask cultures.

A mechanistic model for gas-liquid mass transfer prediction in a rocking disposable bioreactor.

Rocking disposable bioreactors are a newer approach to smaller-scale cell growth that use a cyclic rocking motion to induce mixing and oxygen transfer from the headspace gas into the liquid. Compared with traditional stirred-tank and pneumatic bioreactors, rocking bioreactors operate in a very different physical mode and in this study the oxygen transfer pathways are reassessed to develop a fundamental mass transfer (kL a) model that is compared with experimental data. The model combines two mechanisms, namely surface aeration and oxygenation via a breaking wave with air entrainment, borrowing concepts from ocean wave models. Experimental data for kLa across the range of possible operating conditions (rocking speed, angle, and liquid volume) confirms the validity of the modeling approach, with most predictions falling within ±20% of the experimental values. At low speeds (up to 20 rpm) the surface aeration mechanism is shown to be dominant with a kLa of around 3.5 hr-1 , while at high speeds (40 rpm) and angles the breaking wave mechanism contributes up to 91% of the overall kLa (65 hr-1 ). This model provides an improved fundamental basis for understanding gas-liquid mass transfer for the operation, scale-up, and potential design improvements for rocking bioreactors.

Production of cellulosic butyrate and 3-hydroxybutyrate in engineered Escherichia coli.

Being the most abundant renewable organic substance on Earth, lignocellulosic biomass has acted as an attractive and cost-effective feedstock for biobased production of value-added products. However, lignocellulosic biomass should be properly treated for its effective utilization during biotransformation. The current work aimed to demonstrate biobased production of butyrate and 3-hydroxybutyrate (3-HB) in engineered Escherichia coli using pretreated and detoxified aspen tree (Populus tremuloides) wood chips as the feedstock. Various bioprocessing and genetic/metabolic factors limiting the production of cellulosic butyrate and 3-HB were identified. With these developed bioprocessing strategies and strain engineering approaches, major carbons in the hydrolysate, including glucose, xylose, and even acetate, could be completely dissimilated during shake-flask cultivation with up to 1.68 g L-1 butyrate, 8.95 g L-1 3-HB, and minimal side metabolites (i.e., acetate and ethanol) being obtained. Our results highlight the importance of consolidating bioprocess and genetic engineering strategies for effective biobased production from lignocellulosic biomass.

Strain engineering for microbial production of value-added chemicals and fuels from glycerol.

While the widespread reliance on fossil fuels is driven by their low cost and relative abundance, this fossil-based economy has been deemed unsustainable and, therefore, the adoption of sustainable and environmentally compatible energy sources is on the horizon. Biorefinery is an emerging approach that integrates metabolic engineering, synthetic biology, and systems biology principles for the development of whole-cell catalytic platforms for biomanufacturing. Due to the high degree of reduction and low cost, glycerol, either refined or crude, has been recognized as an ideal feedstock for the production of value-added biologicals, though microbial dissimilation of glycerol sometimes can be difficult particularly under anaerobic conditions. While strain development for glycerol biorefinery is widely reported in the literature, few, if any, commercialized bioprocesses have been developed as a result, such that engineering of glycerol metabolism in microbial hosts remains an untapped opportunity in biomanufacturing. Here we review the recent progress made in engineering microbial hosts for the production of biofuels, diols, organic acids, biopolymers, and specialty chemicals from glycerol. We begin with a broad outline of the major pathways for fermentative and respiratory glycerol dissimilation and key end metabolites, and then focus our analysis on four key genera of bacteria known to naturally dissimilate glycerol, i.e. Klebsiella, Citrobacter, Clostridium, and Lactobacillus, in addition to Escherichia coli, and systematically review the progress made toward engineering these microorganisms for glycerol biorefinery. We also identify the major biotechnological and bioprocessing advantages and disadvantages of each genus, and bottlenecks limiting the production of target metabolites from glycerol in engineered strains. Our analysis culminates in the development of potential strategies to overcome the current technical limitations identified for commonly employed strains, with an outlook on the suitability of different hosts for the production of key metabolites and avenues for their future development into biomanufacturing platforms.

Metabolic engineering of Bacillus subtilis for l-valine overproduction.

Bacillus subtilis has been commonly applied to industrial enzyme production due to its genetic tractability, "generally recognized as safe (GRAS)" status, and robust growth characteristics. In spite of its ideal attributes as a biomanufacturing platform, B. subtilis has seen limited use in the production of other value-added biochemicals. Here, we report the derivation of engineered strains of B. subtilis for l-valine overproduction using our recently developed CRISPR (clustered regularly interspaced palindromic repeats)-Cas9 (CRISPR-associated [protein] 9) toolkit. We first manipulate the native l-valine biosynthetic pathway by relieving transcriptional and allosteric regulation, resulting in a >14-fold increase in the l-valine titer, compared to the wild-type strain. We subsequently identify and eliminate factors limiting l-valine overproduction, specifically increasing pyruvate availability and blocking the competing l-leucine and l-isoleucine biosynthetic pathways. By inactivating (a) pdhA, encoding the E1α subunit of the pyruvate dehydrogenase complex, to increase the intracellular pyruvate pool, and (b) leuA and ilvA, respectively encoding 2-isopropylmalate synthase and l-threonine dehydratase, to abolish the competing pathways, the l-valine titer reached 4.61 g/L in shake flask cultures. Our engineered l-valine-overproducing strains of B. subtilis are devoid of plasmids and do not sporulate due to the inactivation of sigF, encoding the sporulation-specific transcription factor σ F , making them attractive for large-scale l-valine production. However, acetate dissimilation was identified as limiting l-valine overproduction in ΔpdhA B. subtilis strains, and improving acetate dissimilation or identifying alternate modes of increasing pyruvate pools to enhance l-valine-overproduction should be explored.

Metabolic engineering to enhance heterologous production of hyaluronic acid in Bacillus subtilis.

Hyaluronic acid (HA) is a high-value biopolymer that is produced in large scales using attenuated strains ofgroup C streptococci. However, due to the pathogenicity and fastidious nature of these bacteria, the development of bioprocesses for HA production centered on robust 'Generally Recognized as Safe (GRAS)' organisms, such as Bacillus subtilis, is of increased interest. Here, we report metabolic engineering of novel B. subtilis strains in which the carbon flux has been partially diverted from central metabolism, i.e. the pentose phosphate pathway (PPP) and glycolysis, into HA biosynthesis. First, an improved base strain of B. subtilis was engineered for more effective HA production with less susceptibility to catabolite repression when expressing genes from a xylose-inducible promoter. Subsequently, Clustered Regularly Interspaced Palindromic Repeats interference (CRISPRi) was applied to reduce the expression of individual pfkA or zwf in the base strain, leading to substantial improvements to the HA titer with a concomitant decrease in the molecular weight (MW). On the other hand, multiplexed repression of both pfkA and zwf expression resulted in increases to the HA titer of up to 108% and enhancements to the MW, compared to the base strain. Moreover, the addition of exogenous HA monomers, i.e. glucuronic acid (GlcUA) and N-acetyl-glucosamine (GlcNAc), to B. subtilis cultures markedly improved the HA MW but decreased the HA titer, providing insights into the mechanism of HA biosynthesis by streptococcal hyaluronan synthase (SeHAS) in B. subtilis. Our study demonstrates the successful application of metabolic engineering strategies to establish B. subtilis as an effective platform for high-level HA production.

Application of hydrocarbon and perfluorocarbon oxygen vectors to enhance heterologous production of hyaluronic acid in engineered Bacillus subtilis.

In microbial cultivations for hyaluronic acid (HA) production, oxygen can be a limiting substrate due to its poor solubility in aqueous medium and the substantial increase in culture viscosity at relatively low HA titers. Shear stress due to the high agitation and aeration rates required to overcome oxygen limitation may reduce the quality (i.e., molecular weight) of HA, and production costs associated with power consumption and supplemental oxygen may be excessive. Here, we report the application of oxygen vectors to the heterologous production of HA in engineered Bacillus subtilis, leading to significantly improved culture performance. We first derived an improved HA-producing strain of B. subtilis through engineering of the promoter driving coexpression of seHas and tuaD, leading to high-level HA production. Out of seven potential oxygen vectors evaluated in a preliminary screening, significant improvements to the HA titer and/or cell density were observed in cultures containing n-heptane, n-hexadecane, perfluoromethyldecalin, and perfluoro-1,3-dimethylcyclohexane. Adjustments to the vector concentration, timing of vector addition, and the agitation rate resulted in further enhancements, with the HA titer reaching up to 4.5 g/L after only 10 hr cultivation. Moreover, our results indicate that certain vectors may alter the functional expression of Class I hyaluronan synthase (HAS) in B. subtilis, and that higher shear rates may drive more carbon flux through the HA biosynthetic pathway without negatively affecting the MW. Our study demonstrates the efficacy of oxygen vectors to enhance heterologous HA production in B. subtilis, and provides valuable insight for future bioprocess development in microbial HA production.

Engineering of cell membrane to enhance heterologous production of hyaluronic acid in Bacillus subtilis.

Hyaluronic acid (HA) is a high-value biopolymer used in the biomedical, pharmaceutical, cosmetic, and food industries. Current methods of HA production, including extraction from animal sources and streptococcal cultivations, are associated with high costs and health risks. Accordingly, the development of bioprocesses for HA production centered on robust "Generally Recognized as Safe (GRAS)" organisms such as Bacillus subtilis is highly attractive. Here, we report the development of novel strains of B. subtilis in which the membrane cardiolipin (CL) content and distribution has been engineered to enhance the functional expression of heterologously expressed hyaluronan synthase (HAS) of Streptococcus equisimilis (SeHAS), in turn, improving the culture performance for HA production. Elevation of membrane CL levels via overexpressing components involved in the CL biosynthesis pathway, and redistribution of CL along the lateral membrane via repression of the cell division initiator protein FtsZ resulted in increases to the HA titer of up to 204% and peak molecular weight of up to 2.2 MDa. Moreover, removal of phosphatidylethanolamine and neutral glycolipids from the membrane of HA-producing B. subtilis via inactivation of pssA and ugtP, respectively, has suggested the lipid dependence for functional expression of SeHAS. Our study demonstrates successful application of membrane engineering strategies to develop an effective platform for biomanufacturing of HA with B. subtilis strains expressing Class I streptococcal HAS.

A semi-empirical glycosylation model of a camelid monoclonal antibody under hypothermia cell culture conditions.

The impact of cell culture environment on the glycan distribution of a monoclonal antibody (mAb) has been investigated through a combination of experiments and modeling. A newly developed CHO DUXB cell line was cultivated at two levels of initial Glutamine (Gln) concentrations (0, 4 mM) and incubation temperatures of (33 and 37 °C) in batch operation mode. Hypothermia was applied either through the entire culture duration or only during the post-exponential phase. Beyond reducing cell growth and increasing productivity, hypothermia significantly altered the galactosylation index profiles as compared to control conditions. A novel semi-empirical dynamic model was proposed for elucidating the connections between the extracellular cell culture conditions to galactosylation index. The developed model is based on a simplified balance of nucleotides sugars and on the correlation between sugars' levels to the galactosylation index (GI). The model predictions were found to be in a good agreement with the experimental data. The proposed empirical model is expected to be useful for controlling the glycoprofiles by manipulating culture conditions.

Recent advances in engineering propionyl-CoA metabolism for microbial production of value-added chemicals and biofuels.

Diminishing fossil fuel reserves and mounting environmental concerns associated with petrochemical manufacturing practices have generated significant interests in developing whole-cell biocatalytic systems for the production of value-added chemicals and biofuels. Although acetyl-CoA is a common natural biogenic precursor for the biosynthesis of numerous metabolites, propionyl-CoA is unpopular and non-native to most organisms. Nevertheless, with its C3-acyl moiety as a discrete building block, propionyl-CoA can serve as another key biogenic precursor to several biological products of industrial importance. As a result, engineering propionyl-CoA metabolism, particularly in genetically tractable hosts with the use of inexpensive feedstocks, has paved an avenue for novel biomanufacturing. Herein, we present a systematic review on manipulation of propionyl-CoA metabolism as well as relevant genetic and metabolic engineering strategies for microbial production of value-added chemicals and biofuels, including odd-chain alcohols and organic acids, bio(co)polymers and polyketides. [Formula: see text].

Engineering of Escherichia coli for direct and modulated biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer using unrelated carbon sources.

While poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] is a biodegradable commodity plastic with broad applications, its microbial synthesis is hindered by high production costs primarily associated with the supplementation of related carbon substrates (e.g. propionate or valerate). Here we report construction of engineered Escherichia coli strains for direct synthesis of P(3HB-co-3HV) from an unrelated carbon source (e.g. glucose or glycerol). First, an E. coli strain with an activated sleeping beauty mutase (Sbm) operon was used to generate propionyl-CoA as a precursor. Next, two acetyl-CoA moieties or acetyl-CoA and propionyl-CoA were condensed to form acetoacetyl-CoA and 3-ketovaleryl-CoA, respectively, by functional expression of ß-ketothiolases from Cupriavidus necator (i.e. PhaA and BktB). The resulting thioester intermediates were channeled into the polyhydroxyalkanoate (PHA) biosynthetic pathway through functional expression of acetoacetyl-CoA reductase (PhaB) for thioester reduction and PHA synthase (PhaC) for subsequent polymerization. Metabolic engineering of E. coli host strains was further conducted to enhance total PHA content and the 3-hydroxyvaleryl (3HV) monomer fraction in the copolymer. Using a selection of engineered E. coli strains for batch cultivation with an unrelated carbon source, we achieved high-level P(3HB-co-3HV) production with the 3HV monomer fraction ranging from 3 to 19 mol%, demonstrating the potential industrial applicability of these whole-cell biocatalysts.

Genome-directed analysis of prophage excision, host defence systems, and central fermentative metabolism in Clostridium pasteurianum.

Clostridium pasteurianum is emerging as a prospective host for the production of biofuels and chemicals, and has recently been shown to directly consume electric current. Despite this growing biotechnological appeal, the organism's genetics and central metabolism remain poorly understood. Here we present a concurrent genome sequence for the C. pasteurianum type strain and provide extensive genomic analysis of the organism's defence mechanisms and central fermentative metabolism. Next generation genome sequencing produced reads corresponding to spontaneous excision of a novel phage, designated φ6013, which could be induced using mitomycin C and detected using PCR and transmission electron microscopy. Methylome analysis of sequencing reads provided a near-complete glimpse into the organism's restriction-modification systems. We also unveiled the chief C. pasteurianum Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) locus, which was found to exemplify a Type I-B system. Finally, we show that C. pasteurianum possesses a highly complex fermentative metabolism whereby the metabolic pathways enlisted by the cell is governed by the degree of reductance of the substrate. Four distinct fermentation profiles, ranging from exclusively acidogenic to predominantly alcohologenic, were observed through redox consideration of the substrate. A detailed discussion of the organism's central metabolism within the context of metabolic engineering is provided.

Extending CRISPR-Cas9 Technology from Genome Editing to Transcriptional Engineering in the Genus Clostridium.

The discovery and exploitation of the prokaryotic adaptive immunity system based on clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins have revolutionized genetic engineering. CRISPR-Cas tools have enabled extensive genome editing as well as efficient modulation of the transcriptional program in a multitude of organisms. Progress in the development of genetic engineering tools for the genus Clostridium has lagged behind that of many other prokaryotes, presenting the CRISPR-Cas technology an opportunity to resolve a long-existing issue. Here, we applied the Streptococcus pyogenes type II CRISPR-Cas9 (SpCRISPR-Cas9) system for genome editing in Clostridium acetobutylicum DSM792. We further explored the utility of the SpCRISPR-Cas9 machinery for gene-specific transcriptional repression. For proof-of-concept demonstration, a plasmid-encoded fluorescent protein gene was used for transcriptional repression in C. acetobutylicum Subsequently, we targeted the carbon catabolite repression (CCR) system of C. acetobutylicum through transcriptional repression of the hprK gene encoding HPr kinase/phosphorylase, leading to the coutilization of glucose and xylose, which are two abundant carbon sources from lignocellulosic feedstocks. Similar approaches based on SpCRISPR-Cas9 for genome editing and transcriptional repression were also demonstrated in Clostridium pasteurianum ATCC 6013. As such, this work lays a foundation for the derivation of clostridial strains for industrial purposes. IMPORTANCE: After recognizing the industrial potential of Clostridium for decades, methods for the genetic manipulation of these anaerobic bacteria are still underdeveloped. This study reports the implementation of CRISPR-Cas technology for genome editing and transcriptional regulation in Clostridium acetobutylicum, which is arguably the most common industrial clostridial strain. The developed genetic tools enable simpler, more reliable, and more extensive derivation of C. acetobutylicum mutant strains for industrial purposes. Similar approaches were also demonstrated in Clostridium pasteurianum, another clostridial strain that is capable of utilizing glycerol as the carbon source for butanol fermentation, and therefore can be arguably applied in other clostridial strains.

Development of a CRISPR-Cas9 Tool Kit for Comprehensive Engineering of Bacillus subtilis.

UNLABELLED: The establishment of a clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system for strain construction in Bacillus subtilis is essential for its progression toward industrial utility. Here we outline the development of a CRISPR-Cas9 tool kit for comprehensive genetic engineering in B. subtilis In addition to site-specific mutation and gene insertion, our approach enables continuous genome editing and multiplexing and is extended to CRISPR interference (CRISPRi) for transcriptional modulation. Our tool kit employs chromosomal expression of Cas9 and chromosomal transcription of guide RNAs (gRNAs) using a gRNA transcription cassette and counterselectable gRNA delivery vectors. Our design obviates the need for multicopy plasmids, which can be unstable and impede cell viability. Efficiencies of up to 100% and 85% were obtained for single and double gene mutations, respectively. Also, a 2.9-kb hyaluronic acid (HA) biosynthetic operon was chromosomally inserted with an efficiency of 69%. Furthermore, repression of a heterologous reporter gene was achieved, demonstrating the versatility of the tool kit. The performance of our tool kit is comparable with those of systems developed for Escherichia coli and Saccharomyces cerevisiae, which rely on replicating vectors to implement CRISPR-Cas9 machinery. IMPORTANCE: In this paper, as the first approach, we report implementation of the CRISPR-Cas9 system in Bacillus subtilis, which is recognized as a valuable host system for biomanufacturing. The study enables comprehensive engineering of B. subtilis strains with virtually any desired genotypes/phenotypes and biochemical properties for extensive industrial application.

Disruption of the Reductive 1,3-Propanediol Pathway Triggers Production of 1,2-Propanediol for Sustained Glycerol Fermentation by Clostridium pasteurianum.

UNLABELLED: Crude glycerol, the major by-product of biodiesel production, is an attractive bioprocessing feedstock owing to its abundance, low cost, and high degree of reduction. In line with the advent of the biodiesel industry, Clostridium pasteurianum has gained prominence as a result of its unique capacity to convert waste glycerol into n-butanol, a high-energy biofuel. However, no efforts have been directed at abolishing the production of 1,3-propanediol (1,3-PDO), the chief competing product of C. pasteurianum glycerol fermentation. Here, we report rational metabolic engineering of C. pasteurianum for enhanced n-butanol production through inactivation of the gene encoding 1,3-PDO dehydrogenase (dhaT). In spite of current models of anaerobic glycerol dissimilation, culture growth and glycerol utilization were unaffected in the dhaT disruption mutant (dhaT::Ll.LtrB). Metabolite characterization of the dhaT::Ll.LtrB mutant revealed an 83% decrease in 1,3-PDO production, encompassing the lowest C. pasteurianum 1,3-PDO titer reported to date (0.58 g liter(-1)). With 1,3-PDO formation nearly abolished, glycerol was converted almost exclusively to n-butanol (8.6 g liter(-1)), yielding a high n-butanol selectivity of 0.83 g n-butanol g(-1) of solvents compared to 0.51 g n-butanol g(-1) of solvents for the wild-type strain. Unexpectedly, high-performance liquid chromatography (HPLC) analysis of dhaT::Ll.LtrB mutant culture supernatants identified a metabolite peak consistent with 1,2-propanediol (1,2-PDO), which was confirmed by nuclear magnetic resonance (NMR). Based on these findings, we propose a new model for glycerol dissimilation by C. pasteurianum, whereby the production of 1,3-PDO by the wild-type strain and low levels of both 1,3-PDO and 1,2-PDO by the engineered mutant balance the reducing equivalents generated during cell mass synthesis from glycerol. IMPORTANCE: Organisms from the genus Clostridium are perhaps the most notable native cellular factories, owing to their vast substrate utilization range and equally diverse variety of metabolites produced. The ability of C. pasteurianum to sustain redox balance and glycerol fermentation despite inactivation of the 1,3-PDO pathway is a testament to the exceptional metabolic flexibility exhibited by clostridia. Moreover, identification of a previously unknown 1,2-PDO-formation pathway, as detailed herein, provides a deeper understanding of fermentative glycerol utilization in clostridia and will inform future metabolic engineering endeavors involving C. pasteurianum To our knowledge, the C. pasteurianum dhaT disruption mutant derived in this study is the only organism that produces both 1,2- and 1,3-PDOs. Most importantly, the engineered strain provides an excellent platform for highly selective production of n-butanol from waste glycerol.