Inactivation of the Levansucrase Gene in Paenibacillus polymyxa DSM 365 Diminishes Exopolysaccharide Biosynthesis during 2,3-Butanediol Fermentation.

The formation of exopolysaccharides (EPSs) during 2,3-butanediol (2,3-BD) fermentation by Paenibacillus polymyxa increases medium viscosity, which in turn presents considerable technical and economic challenges to 2,3-BD downstream processing. To eliminate EPS production during 2,3-BD fermentation, we used homologous recombination to disable the EPS biosynthetic pathway in P. polymyxa The gene which encodes levansucrase, the major enzyme responsible for EPS biosynthesis in P. polymyxa, was successfully disrupted. The P. polymyxa levansucrase null mutant produced 2.5 ± 0.1 and 1.2 ± 0.2 g/liter EPS on sucrose and glucose, respectively, whereas the wild type produced 21.7 ± 2.5 and 3.1 ± 0.0 g/liter EPS on the same substrates, respectively. These levels of EPS translate to 8.7- and 2.6-fold decreases in EPS formation by the levansucrase null mutant on sucrose and glucose, respectively, relative to that by the wild type, with no significant reduction in 2,3-BD production. Inactivation of EPS biosynthesis led to a considerable increase in growth. On glucose and sucrose, the cell biomass of the levansucrase null mutant (8.1 ± 0.8 and 6.5 ± 0.3 g/liter, respectively) increased 1.4-fold compared to that of the wild type (6.0 ± 0.1 and 4.6 ± 0.3 g/liter, respectively) grown on the same substrates. Evaluation of the genetic stability of the levansucrase null mutant showed that it remained genetically stable over fifty generations, with no observable decrease in growth or 2,3-BD formation, with or without antibiotic supplementation. Hence, the P. polymyxa levansucrase null mutant has potential for use as an industrial biocatalyst for a cost-effective large-scale 2,3-BD fermentation process devoid of EPS-related challenges.IMPORTANCE Given the current barrage of attention and research investments toward the production of next-generation fuels and chemicals, of which 2,3-butanediol (2,3-BD) produced by nonpathogenic Paenibacillus species is perhaps one of the most vigorously pursued, tools for engineering Paenibacillus species are intensely sought after. Exopolysaccharide (EPS) production during 2,3-BD fermentation constitutes a problem during downstream processing. Specifically, EPS negatively impacts 2,3-BD separation from the fermentation broth, thereby increasing the overall cost of 2,3-BD production. The results presented here demonstrate that inactivation of the levansucrase gene in P. polymyxa leads to diminished EPS accumulation. Additionally, a new method for an EPS assay and a simple protocol employing protoplasts for enhanced transformation of P. polymyxa were developed. Overall, although our study shows that levan is not the only EPS produced by P. polymyxa, it represents a significant first step toward developing cost-effective 2,3-BD fermentation devoid of EPS-associated complications during downstream processing.

Effects of Clostridium beijerinckii and Medium Modifications on Acetone-Butanol-Ethanol Production From Switchgrass.

The presence of lignocellulose-derived microbial inhibitory compounds (LDMICs) in lignocellulosic biomass (LB) hydrolysates is a barrier to efficient conversion of LB hydrolysates to fuels and chemicals by fermenting microorganisms. Results from this study provide convincing evidence regarding the effectiveness of metabolically engineered C. beijerinckii NCIMB 8052 for the fermentation of LB-derived hydrolysates to acetone-butanol-ethanol (ABE). The engineered microbial strain (C. beijerinckii_SDR) was produced by the integration of an additional copy of a short-chain dehydrogenase/reductase (SDR) gene (Cbei_3904) into the chromosome of C. beijerinckii NCIMB 8052 wildtype, where it is controlled by the constitutive thiolase promoter. The C. beijerinckii_SDR and C. beijerinckii NCIMB 8052 wildtype were used for comparative fermentation of non-detoxified and detoxified hydrothermolysis-pretreated switchgrass hydrolysates (SHs) with and without (NH4)2CO3 supplementation. In the absence of (NH4)2CO3, fermentation of non-detoxified SH with C. beijerinckii_SDR resulted in the production of 3.13- and 2.25-fold greater quantities of butanol (11.21 g/L) and total ABE (20.24 g/L), respectively, than the 3.58 g/L butanol and 8.98 g/L ABE produced by C. beijerinckii_wildtype. When the non-detoxified SH was supplemented with (NH4)2CO3, concentrations were similar for butanol (9.5 compared with 9.2 g/L) and ABE (14.2 compared with 13.5 g/L) produced by C. beijerinckii_SDR and C. beijerinckii_wildtype, respectively. Furthermore, when C. beijerinckii_SDR and C. beijerinckii_wildtype were cultured in detoxified SH medium, C. beijerinckii_SDR produced 1.11- and 1.18-fold greater quantities of butanol and ABE, respectively, than when there was culturing with C. beijerinckii_wildtype. When the combined results of the present study are considered, conclusions are that the microbial strain and medium modifications of the fermentation milieu resulted in greater production of fuels and chemicals from non-detoxified LB hydrolysates.

Ribozyme-Mediated Downregulation Uncovers DNA Integrity Scanning Protein A (DisA) as a Solventogenesis Determinant in Clostridium beijerinckii.

Carbon catabolite repression (CCR) limits microbial utilization of lignocellulose-derived pentoses. To relieve CCR in Clostridium beijerinckii NCIMB 8052, we sought to downregulate catabolite control protein A (CcpA) using the M1GS ribozyme technology. A CcpA-specific ribozyme was constructed by tethering the catalytic subunit of Escherichia coli RNase P (M1 RNA) to a guide sequence (GS) targeting CcpA mRNA (M1GSCcpA). As negative controls, the ribozyme M1GSCcpA-Sc (constructed with a scrambled GSCcpA) or the empty plasmid pMTL500E were used. With a ∼3-fold knockdown of CcpA mRNA in C. beijerinckii expressing M1GSCcpA (C. beijerinckii_M1GSCcpA) relative to both controls, a modest enhancement in mixed-sugar utilization and solvent production was achieved. Unexpectedly, C. beijerinckii_M1GSCcpA-Sc produced 50% more solvent than C. beijerinckii_pMTL500E grown on glucose + arabinose. Sequence complementarity (albeit suboptimal) suggested that M1GSCcpA-Sc could target the mRNA encoding DNA integrity scanning protein A (DisA), an expectation that was confirmed by a 53-fold knockdown in DisA mRNA levels. Therefore, M1GSCcpA-Sc was renamed M1GSDisA. Compared to C. beijerinckii_M1GSCcpA and _pMTL500E, C. beijerinckii_M1GSDisA exhibited a 7-fold decrease in the intracellular c-di-AMP level after 24 h of growth and a near-complete loss of viability upon exposure to DNA-damaging antibiotics. Alterations in c-di-AMP-mediated signaling and cell cycling likely culminate in a sporulation delay and the solvent production gains observed in C. beijerinckii_M1GSDisA. Successful knockdown of the CcpA and DisA mRNAs demonstrate the feasibility of using M1GS technology as a metabolic engineering tool for increasing butanol production in C. beijerinckii.

Use of Spondias Mombin fruit pulp as a substrate for biosurfactant production.

In this study, we explored the possibility of utilizing the succulent pulp of Spondias mombin (SM) as feedstock for the synthesis of biosurfactants by Pseudomonas spp. The cultures were composed of basic mineral medium amended with SM, SM + glucose, glucose (GLC), and nutrient broth (NB) as carbon sources. Biosurfactant production was determined by surface-active properties such as hemolysis, emulsification index (E24), drop collapse, oil-spreading assays, and reduction of surface tension. The stability of the biosurfactants was monitored across different temperature and pH regimes while chemical components of the extracted biosurfactants were determined by thin-layer chromatography. Biosurfactants synthesized from SM as sole substrate showed the highest emulsification index (56.35%), oil-spreading capacity (4.4 ± 1.31 cm), hemolysis (3.10 ± 0.02 cm), the shortest time for drop collapse (30 s), and surface tension reduction (24 mN/m). Biosurfactant concentrations ranged from 0.07 ± 0.01 in the NB to 2.08 ± 0.01 g/L in the media amended with SM. Chemical characterization revealed significant concentrations of carbohydrates and lipids in the biosurfactant produced from SM (1.2 ± 0.17 and 0.88 ± 0.04 g/L, respectively) when compared to SM + glucose (0.92 ± 0.05, and 0.62 ± 0.02 g/L, respectively), glucose (0.35 ± 0.04 and 0.13 ± 0.02 g/L, respectively), and nutrient broth (0.06 ± 0.03 and 0.01 ± 0.01 g/L, respectively). The biosurfactants were stable over a wide range of temperature while E24 increased with pH. Our results show the viability of SM fruit pulp as low-cost feedstock for industrial-scale production of biosurfactants using Pseudomonas spp.

Chromosomal integration of aldo-keto-reductase and short-chain dehydrogenase/reductase genes in Clostridium beijerinckii NCIMB 8052 enhanced tolerance to lignocellulose-derived microbial inhibitory compounds.

In situ detoxification of lignocellulose-derived microbial inhibitory compounds is an economical strategy for the fermentation of lignocellulose-derived sugars to fuels and chemicals. In this study, we investigated homologous integration and constitutive expression of Cbei_3974 and Cbei_3904, which encode aldo-keto reductase and previously annotated short chain dehydrogenase/reductase, respectively, in Clostridium beijerinckii NCIMB 8052 (Cb), resulting in two strains: Cb_3974 and Cb_3904. Expression of Cbei_3974 led to 2-fold increase in furfural detoxification relative to Cb_3904 and Cb_wild type. Correspondingly, butanol production was up to 1.2-fold greater in furfural-challenged cultures of Cb_3974 relative to Cb_3904 and Cb_wild type. With 4-hydroxybezaldehyde and syringaldehyde supplementation, Cb_3974 showed up to 2.4-fold increase in butanol concentration when compared to Cb_3904 and Cb_wild type. Syringic and vanillic acids were considerably less deleterious to all three strains of Cb tested. Overall, Cb_3974 showed greater tolerance to furfural, 4-hydroxybezaldehyde, and syringaldehyde with improved capacity for butanol production. Hence, development of Cb_3974 represents a significant progress towards engineering solventogenic Clostridium species that are tolerant to lignocellulosic biomass hydrolysates as substrates for ABE fermentation.

Investigation of relationship between 2,3-butanediol toxicity and production during growth of Paenibacillus polymyxa.

Understanding the capacity of Paenibacillus polymyxa DSM 365 to tolerate increasing concentrations of 2,3-butanediol (2,3-BD) is critical to engineering a 2,3-BD-overproducing strain. Hence, we investigated the response of P. polymyxa to high 2,3-BD concentrations. In fed-batch cultures (6-L bioreactor) 2,3-BD was accumulated to a maximum concentration of 47g/L despite the presence of residual 13g/L glucose in the medium. Concomitantly, accumulation of acetoin, the precursor of 2,3-BD increased after maximum 2,3-BD concentration was reached, suggesting that 2,3-BD was reconverted to acetoin after the concentration tolerance threshold of 2,3-BD was exceeded. Cultures of P. polymyxa were then challenged with levo-2,3-BD (20, 40 and 60g/L) at 0h in a glucose medium, and a concentration dependent growth inhibition response to levo-2,3-BD was observed. The growth of P. polymyxa was completely inhibited by 60g/L levo-2,3-BD. Furthermore, P. polymyxa was challenged with incremental 2,3-BD concentrations (20, 40 and 60g/L at 12, 24 and 36h, respectively) to mimic 2,3-BD accumulation during fermentation. Interestingly, 2,3-BD was reconverted to acetoin when its concentration reached 60g/L, possibly to alleviate 2,3-BD toxicity. Collectively, our findings indicate that 2,3-BD-mediated toxicity is a major metabolic impediment to 2,3-BD overproduction, thus, making it an important metabolic engineering target towards rational design of a 2,3-BD-overproducing strain.