Agricultural Waste Management by Production of Second-Generation Bioethanol from Sugarcane Bagasse Using Indigenous Yeast Strain.

In the wake of rapid industrialization and burgeoning transportation networks, the escalating demand for fossil fuels has accelerated the depletion of finite energy reservoirs, necessitating urgent exploration of sustainable alternatives. To address this, current research is focusing on renewable fuels like second-generation bioethanol from agricultural waste such as sugarcane bagasse. This approach not only circumvents the contentious issue of food-fuel conflicts associated with biofuels but also tackles agricultural waste management. In the present study indigenous yeast strain, Clavispora lusitaniae QG1 (MN592676), was isolated from rotten grapes to ferment xylose sugars present in the hemicellulose content of sugarcane bagasse. To liberate the xylose sugars, dilute acid pretreatment was performed. The highest reducing sugars yield was 1.2% obtained at a temperature of 121 °C for 15 min, a solid-to-liquid ratio of 1:25 (% w/v), and an acid concentration of 1% dilute acid H2SO4 that was significantly higher (P < 0.001) yield obtained under similar conditions at 100 °C for 1 h. The isolated strain was statistically optimized for fermentation process by Plackett-Burman design to achieve the highest ethanol yield. Liberated xylose sugars were completely utilized by Clavispora lusitaniae QG1 (MN592676) and gave 100% ethanol yield. This study optimizes both fermentation process and pretreatment of sugarcane bagasse to maximize bioethanol yield and demonstrates the ability of isolated strain to effectively utilize xylose as a carbon source. The desirable characteristics depicted by strain Clavispora lusitaniae shows its promising utilization in management of industrial waste like sugarcane bagasse by its conversion into renewable biofuels like bioethanol.

Utilization of microalgae for agricultural runoff remediation and sustainable biofuel production through an integrated biorefinery approach.

Generally wastewater such agricultural runoff is considered a nuisance; however, it could be harnessed as a potential source of nutrients like nitrates and phosphates in integrated biorefinery context. In the current study, microalgae Chlorella sp. S5 was used for bioremediation of agricultural runoff and the leftover algal biomass was used as a potential source for production of biofuels in an integrated biorefinery context. The microalgae Chlorella sp. S5 was cultivated on Blue Green (BG 11) medium and a comprehensive optimization of different parameters including phosphates, nitrates, and pH was carried out to acquire maximum algal biomass enriched with high lipids content. Dry biomass was quantified using the solvent extraction technique, while the identification of nitrates and phosphates in agricultural runoff was carried out using commercial kits. The algal extracted lipids (oils) were employed in enzymatic trans-esterification for biodiesel production using whole-cell biomass of Bacillus subtilis Q4 MZ841642. The resultant fatty acid methyl esters (FAMEs) were analyzed using Fourier transform infrared (FTIR) spectroscopy and gas chromatography coupled with mass spectrometry (GC-MS). Subsequently, both the intact algal biomass and its lipid-depleted algal biomass were used for biogas production within a batch anaerobic digestion setup. Interestingly, Chlorella sp. S5 demonstrated a substantial reduction of 95% in nitrate and 91% in phosphate from agricultural runoff. The biodiesel derived from algal biomass exhibited a noteworthy total FAME content of 98.2%, meeting the quality standards set by American Society for Testing and Materials (ASTM) and European union (EU) standards. Furthermore, the biomethane yields obtained from whole biomass and lipid-depleted biomass were 330.34 NmL/g VSadded and 364.34 NmL/g VSadded, respectively. In conclusion, the findings underscore the potent utility of Chlorella sp. S5 as a multi-faceted resource, proficiently employed in a sequential cascade for treating agricultural runoff, producing biodiesel, and generating biogas within the integrated biorefinery concept.

Development of genetic tools for heterologous protein expression in a pentose-utilizing environmental isolate of Pseudomonas putida.

Pseudomonas putida has emerged as a promising host for the conversion of biomass-derived sugars and aromatic intermediates into commercially relevant biofuels and bioproducts. Most of the strain development studies previously published have focused on P. putida KT2440, which has been engineered to produce a variety of non-native bioproducts. However, P. putida is not capable of metabolizing pentose sugars, which can constitute up to 25% of biomass hydrolysates. Related P. putida isolates that metabolize a larger fraction of biomass-derived carbon may be attractive as complementary hosts to P. putida KT2440. Here we describe genetic tool development for P. putida M2, a soil isolate that can metabolize pentose sugars. The functionality of five inducible promoter systems and 12 ribosome binding sites was assessed to regulate gene expression. The utility of these expression systems was confirmed by the production of indigoidine from C6 and C5 sugars. Chromosomal integration and expression of non-native genes was achieved by using chassis-independent recombinase-assisted genome engineering (CRAGE) for single-step gene integration of biosynthetic pathways directly into the genome of P. putida M2. These genetic tools provide a foundation to develop hosts complementary to P. putida KT2440 and expand the ability of this versatile microbial group to convert biomass to bioproducts.

Revealing oxidative pentose metabolism in new Pseudomonas putida isolates.

The Pseudomonas putida group in the Gammaproteobacteria has been intensively studied for bioremediation and plant growth promotion. Members of this group have recently emerged as promising hosts to convert intermediates derived from plant biomass to biofuels and biochemicals. However, most strains of P. putida cannot metabolize pentose sugars derived from hemicellulose. Here, we describe three isolates that provide a broader view of the pentose sugar catabolism in the P. putida group. One of these isolates clusters with the well-characterized P. alloputida KT2440 (Strain BP6); the second isolate clustered with plant growth-promoting strain P. putida W619 (Strain M2), while the third isolate represents a new species in the group (Strain BP8). Each of these isolates possessed homologous genes for oxidative xylose catabolism (xylDXA) and a potential xylonate transporter. Strain M2 grew on arabinose and had genes for oxidative arabinose catabolism (araDXA). A CRISPR interference (CRISPRi) system was developed for strain M2 and identified conditionally essential genes for xylose growth. A glucose dehydrogenase was found to be responsible for initial oxidation of xylose and arabinose in strain M2. These isolates have illuminated inherent diversity in pentose catabolism in the P. putida group and may provide alternative hosts for biomass conversion.

Metabolic Engineering of Corynebacterium glutamicum for Production of UDP-N-Acetylglucosamine.

Uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) is an acetylated amino sugar nucleotide that naturally serves as precursor in bacterial cell wall synthesis and is involved in prokaryotic and eukaryotic glycosylation reactions. UDP-GlcNAc finds application in various fields including the production of oligosaccharides and glycoproteins with therapeutic benefits. At present, nucleotide sugars are produced either chemically or in vitro by enzyme cascades. However, chemical synthesis is complex and non-economical, and in vitro synthesis requires costly substrates and often purified enzymes. A promising alternative is the microbial production of nucleotide sugars from cheap substrates. In this study, we aimed to engineer the non-pathogenic, Gram-positive soil bacterium Corynebacterium glutamicum as a host for UDP-GlcNAc production. The native glmS, glmU, and glmM genes and glmM of Escherichia coli, encoding the enzymes for UDP-GlcNAc synthesis from fructose-6-phosphate, were over-expressed in different combinations and from different plasmids in C. glutamicum GRS43, which lacks the glucosamine-6-phosphate deaminase gene (nagB) for glucosamine degradation. Over-expression of glmS, glmU and glmM, encoding glucosamine-6-phosphate synthase, the bifunctional glucosamine-1-phosphate acetyltransferase/N-acetyl glucosamine-1-phosphate uridyltransferase and phosphoglucosamine mutase, respectively, was confirmed using activity assays or immunoblot analysis. While the reference strain C. glutamicum GlcNCg1 with an empty plasmid in the exponential growth phase contained intracellularly only about 0.25 mM UDP-GlcNAc, the best engineered strain GlcNCg4 accumulated about 14 mM UDP-GlcNAc. The extracellular UDP-GlcNAc concentrations in the exponential growth phase did not exceed 2 mg/L. In the stationary phase, about 60 mg UDP-GlcNAc/L was observed extracellularly with strain GlcNCg4, indicating the potential of C. glutamicum to produce and to release the activated sugar into the culture medium. To our knowledge, the observed UDP-GlcNAc levels are the highest obtained with microbial hosts, emphasizing the potential of C. glutamicum as a suitable platform for activated sugar production.

Functional insights into Mycobacterium tuberculosis DevR-dependent transcriptional machinery utilizing Escherichia coli.

DevR/DosR response regulator is believed to participate in virulence, dormancy adaptation and antibiotic tolerance mechanisms of Mycobacterium tuberculosis by regulating the expression of the dormancy regulon. We have previously shown that the interaction of DevR with RNA polymerase is essential for the expression of DevR-regulated genes. Here, we developed a M. tuberculosis-specific in vivo transcription system to enrich our understanding of DevR-RNA polymerase interaction. This in vivo assay involves co-transforming E. coli with two plasmids that express α, ß, ß' and σA subunits of M. tuberculosis RNA polymerase and a third plasmid that harbors a DevR expression cassette and a GFP reporter gene under the DevR-regulated fdxA promoter. We show that DevR-dependent transcription is sponsored exclusively by M. tuberculosis RNA polymerase and regulated by α and σA subunits of M. tuberculosis RNA polymerase. Using this E. coli triple plasmid system to express mutant variants of M. tuberculosis RNA polymerase, we identified E280 residue in C-terminal domain of α and K513 and R515 residues of σA to participate in DevR-dependent transcription. In silico modeling of a ternary complex of DevR, σA domain 4 and fdxA promoter suggest an interaction of Q505, R515 and K513 residues of σA with E178 and D172 residues of DevR and E471 of σA, respectively. These findings provide us with new insights into the interactions between DevR and RNA polymerase of M. tuberculosis which can be targeted for intercepting DevR function. Finally, we demonstrate the utility of this system for screening of anti-DevR compounds.

Development of dual-inducible duet-expression vectors for tunable gene expression control and CRISPR interference-based gene repression in Pseudomonas putida KT2440.

The development of P. putida as an industrial host requires a sophisticated molecular toolbox for strain improvement, including vectors for gene expression and repression. To augment existing expression plasmids for metabolic engineering, we developed a series of dual-inducible duet-expression vectors for P. putida KT2440. A number of inducible promoters (Plac , Ptac , PtetR/tetA and Pbad ) were used in different combinations to differentially regulate the expression of individual genes. Protein expression was evaluated by measuring the fluorescence of reporter proteins (GFP and RFP). Our experiments demonstrated the use of compatible plasmids, a useful approach to coexpress multiple genes in P. putida KT2440. These duet vectors were modified to generate a fully inducible CRISPR interference system using two catalytically inactive Cas9 variants from S. pasteurianus (dCas9) and S. pyogenes (spdCas9). The utility of developed CRISPRi system(s) was demonstrated by repressing the expression of nine conditionally essential genes, resulting in growth impairment and prolonged lag phase for P. putida KT2440 growth on glucose. Furthermore, the system was shown to be tightly regulated, tunable and to provide a simple way to identify essential genes with an observable phenotype.

Construction of a novel dual-inducible duet-expression system for gene (over)expression in Pseudomonas putida.

Pseudomonas putida is a widely used host for metabolic engineering and synthetic biology. However, the use of P. putida has been hampered by the availability of a limited set of expression vectors for producing heterologous proteins. To widen the scope of expression vectors for gene co-expression studies, a previously established dual-inducible expression vector pRG_Duet2 developed for Corynebacterium glutamicum has been modified for use in P. putida. This expression vector, named pRGPDuo2, harbors two origins of replication, colE1 for replication in E. coli and pRO1600 for replication in P. putida. Two multiple cloning sites (MCS1 and MCS2) in pRGPDuo2 are individually controlled by inducible promoters Ptac or PtetR/tetA. Functional validation of pRGPDuo2 was confirmed by the co-expression of genes for the fluorescent proteins namely, superfolder green fluorescent protein (sfGFP), and red fluorescent protein (RFP). Moreover, the strength of the fluorescence signal was dependent on the inducer concentrations present in the culture medium. The expression vector pRGPDuo2 is an attractive addition to the existing repertoire of expression plasmids for expression profiling and adds to the tools available for P. putida metabolic engineering.

A simple dual-inducible CRISPR interference system for multiple gene targeting in Corynebacterium glutamicum.

The development of CRISPR interference (CRISPRi) technology has dramatically increased the pace and the precision of target identification during platform strain development. In order to develop a simple, reliable, and dual-inducible CRISPRi system for the industrially relevant Corynebacterium glutamicum, we combined two different inducible repressor systems in a single plasmid to separately regulate the expression of dCas9 (anhydro-tetracycline-inducible) and a given single guide RNA (IPTG-inducible). The functionality of the resulting vector was demonstrated by targeting the l-arginine biosynthesis pathway in C. glutamicum. By co-expressing dCas9 and a specific single guide RNA targeting the 5'-region of the argininosuccinate lyase gene argH, the specific activity of the target enzyme was down-regulated and in a l-arginine production strain, l-arginine formation was shifted towards citrulline formation. The system was also employed for down-regulation of multiple genes by concatenating sgRNA sequences encoded on one plasmid. Simultaneous down-regulated expression of both argH and the phosphoglucose isomerase gene pgi proved the potential of the system for multiplex targeting. The system can be a promising tool for further pathway engineering in C. glutamicum. Cumulative effects on targeted genes can be rapidly evaluated avoiding tedious and time-consuming traditional gene knockout approaches.

A step forward: Compatible and dual-inducible expression vectors for gene co-expression in Corynebacterium glutamicum.

The Gram-positive bacterium Corynebacterium glutamicum represents a promising platform for the production of amino acids, organic acids, and other bio-products. However, the availability of only few expression vectors limits its use for production purposes, using metabolic engineering approaches when co-expression of several target genes is desired. To widen the scope for co-expression, the pCG1/p15A and pBL1/colE1 replicons were employed to construct the two differentially-inducible and compatible expression vectors pRG_Duet1 and pRG_Duet2. To functionally validate these newly constructed expression vectors, target genes for easily measurable enzymes were cloned and over-expression of these genes was investigated using respective enzyme assays. Furthermore, functionality and co-existence of the pCG1-based C. glutamicum - E. coli shuttle vector pRG_Duet1 were confirmed with pBL1-based expression vectors pRG_Duet2 and pEKEx2, using co-transformation and enzyme assays. The novel shuttle expression vectors pRG_Duet1 and pRG_Duet2 are attractive additions to the existing set of vectors for co-expression studies and metabolic engineering of C. glutamicum.

High level in vivo mucin-type glycosylation in Escherichia coli.

BACKGROUND: Increasing efforts have been made to assess the potential of Escherichia coli strains for the production of complex recombinant proteins. Since a considerable part of therapeutic proteins are glycoproteins, the lack of the post-translational attachment of sugar moieties in standard E. coli expression strains represents a major caveat, thus limiting the use of E. coli based cell factories. The establishment of an E. coli expression system capable of protein glycosylation could potentially facilitate the production of therapeutics with a putative concomitant reduction of production costs. RESULTS: The previously established E. coli strain expressing the soluble form of the functional human-derived glycosyltransferase polypeptide N-acetylgalactosaminyltransferase 2 (GalNAc-T2) was further modified by co-expressing the UDP-GlcNAc 4-epimerase WbgU derived from Plesiomonas shigelloides. This enables the conversion of uridine 5'-diphospho-N-acetylglucosamine (UDP-GlcNAc) to the sugar donor uridine 5'-diphospho-N-acetylgalactosamine (UDP-GalNAc) in the bacterial cytoplasm. Initially, the codon-optimised gene wbgU was inserted into a pET-derived vector and a Tobacco Etch Virus (TEV) protease cleavable polyhistidine-tag was translationally fused to the C- terminus of the amino acid sequence. The 4-epimerase was subsequently expressed and purified. Following the removal of the polyhistidine-tag, WbgU was analysed by circular dichroism spectroscopy to determine folding state and thermal transitions of the protein. The in vitro activity of WbgU was validated by employing a modified glycosyltransferase assay. The conversion of UDP-GlcNAc to UDP-GalNAc was shown by capillary electrophoresis analysis. Using a previously established chaperone pre-/co- expression platform, the in vivo activity of both glycosyltransferase GalNAc-T2 and 4-epimerase WbgU was assessed in E. coli, in combination with a mucin 10-derived target protein. Monitoring glycosylation by liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS), the results clearly indicated the in vivo glycosylation of the mucin-derived acceptor peptide. CONCLUSION: In the present work, the previously established E. coli- based expression system was further optimized and the potential for in vivo O-glycosylation was shown by demonstrating the transfer of sugar moieties to a mucin-derived acceptor protein. The results offer the possibility to assess the practical use of the described expression platform for in vivo glycosylations of important biopharmaceutical compounds in E. coli.

The RamA regulon: complex regulatory interactions in relation to central metabolism in Corynebacterium glutamicum.

Corynebacterium glutamicum is an industrial workhorse used for the production of amino acids and a variety of other chemicals and fuels. Within its regulatory repertoire, C. glutamicum possesses RamA which was initially identified as essential transcriptional regulator of acetate metabolism. Further studies revealed its relevance for ethanol and propionate catabolism and also identified RamA to function as global regulator in the metabolism of C. glutamicum. Thereby, RamA acts as transcriptional activator or repressor of genes encoding enzymes which are involved in carbon uptake, central carbon metabolism, and cell wall synthesis. RamA controls the expression of target genes either directly and/or indirectly by constituting feed-forward loop type of transcriptional motifs with other regulators such as GlxR, SugR, RamB, and GntR1. In this review, we summarize the current knowledge on RamA, its regulon, and its regulatory interplay with other transcriptional regulators coordinating the metabolism of C. glutamicum.