Isobutanol production by combined in vivo and in vitro metabolic engineering.

The production of the biofuel, isobutanol, in E. coli faces limitations due to alcohol toxicity, product inhibition, product recovery, and long-term industrial feasibility. Here we demonstrate an approach of combining both in vivo with in vitro metabolic engineering to produce isobutanol. The in vivo production of α-ketoisovalerate (KIV) was conducted through CRISPR mediated integration of the KIV pathway in bicistronic design (BCD) in E. coli and inhibition of competitive valine pathway using CRISPRi technology. The subsequent in vitro conversion to isobutanol was carried out with engineered enzymes for 2-ketoacid decarboxylase (KIVD) and alcohol dehydrogenase (ADH). For the in vivo production of KIV and subsequent in vitro production of isobutanol, this two-step serial approach resulted in yields of 56% and 93%, productivities of 0.62 and 0.074 g L-1 h-1, and titers of 5.6 and 1.78 g L-1, respectively. Thus, this combined biosynthetic system can be used as a modular approach for producing important metabolites, like isobutanol, without the limitations associated with in vivo production using a consolidated bioprocess.

Deletion of pgi gene in E. coli increases tolerance to furfural and 5-hydroxymethyl furfural in media containing glucose-xylose mixture.

BACKGROUND: Furfural and 5-hydroxymethyl furfural (5-HMF) are key furan inhibitors that are generated due to breakdown of lignocellulosic sugars at high temperature and acidic treatment conditions. Both furfural and 5-HMF act in a synergistic manner to inhibit microbial metabolism and resistance to both is a desirable characteristic for efficient conversion of lignocellulosic carbon to ethanol. Genetic manipulations targeted toward increasing cellular NADPH pools have successfully imparted tolerance against furfural and 5-HMF. In present study, deletion of pgi gene as a strategy to augment carbon flow through pentose phosphate pathway (PPP) was studied in ethanologenic Escherichia coli strain SSK101 to impart tolerance towards either furfural or 5-HMFor both inhibitors together. RESULTS: A key gene of EMP pathway, pgi, was deleted in an ethanologenic E. coli strain SSK42 to yield strain SSK101. In presence of 1 g/L furfural in minimal AM1 media, the rate of biomass formation for strain SSK101 was up to 1.9-fold higher as compared to parent SSK42 strain, and it was able to clear furfural in half the time. Tolerance to inhibitor was associated with glucose as carbon source and not xylose, and the tolerance advantage of SSK101 was neutralized in LB media. Bioreactor studies were performed under binary stress of furfural and 5-HMF (1 g/L each) and different glucose concentrations in a glucose-xylose mixture with final sugar concentration of 5.5%, mimicking major components of dilute acid treated biomass hydrolysate. In the mixture having 6 g/L and 12 g/L glucose, SSK101 strain produced ~ 18 g/L and 20 g/L ethanol, respectively. Interestingly, the maximum ethanol productivity was better at lower glucose load with 0.46 g/(L.h) between 96 and 120 h, as compared to higher glucose load where it was 0.33 g/(L.h) between 144 and 168 h. Importantly, parent strain SSK42 did not exhibit significant metabolic activity under similar conditions of inhibitor load and sugar concentration. CONCLUSIONS: E. coli strain SSK101 with pgi deletion had enhanced tolerance against both furfural and 5-HMF, which was associated with presence of glucose in media. Strain SSK101 also had improved fermentation characteristics under both hyperosmotic as well as binary stress of furfural and 5-HMF in media containing glucose-xylose mixture.

Mining the biomass deconstructing capabilities of rice yellow stem borer symbionts.

BACKGROUND: Efficient deconstruction of lignocellulosic biomass into simple sugars in an economically viable manner is a prerequisite for its global acceptance as a feedstock in bioethanol production. This is achieved in nature by suites of enzymes with the capability of efficiently depolymerizing all the components of lignocellulose. Here, we provide detailed insight into the repertoire of enzymes produced by microorganisms enriched from the gut of the crop pathogen rice yellow stem borer (Scirpophaga incertulas). RESULTS: A microbial community was enriched from the gut of the rice yellow stem borer for enhanced rice straw degradation by sub-culturing every 10 days, for 1 year, in minimal medium with rice straw as the main carbon source. The enriched culture demonstrated high cellulolytic and xylanolytic activity in the culture supernatant. Metatranscriptomic and metaexoproteomic analysis revealed a large array of enzymes potentially involved in rice straw deconstruction. The consortium was found to encode genes ascribed to all five classes of carbohydrate-active enzymes (GHs, GTs, CEs, PLs, and AAs), including carbohydrate-binding modules (CBMs), categorized in the carbohydrate-active enzymes (CAZy) database. The GHs were the most abundant class of CAZymes. Predicted enzymes from these CAZy classes have the potential to digest each cell-wall components of rice straw, i.e., cellulose, hemicellulose, pectin, callose, and lignin. Several identified CAZy proteins appeared novel, having an unknown or hypothetical catalytic counterpart with a known class of CBM. To validate the findings, one of the identified enzymes that belong to the GH10 family was functionally characterized. The enzyme expressed in E. coli efficiently hydrolyzed beechwood xylan, and pretreated and untreated rice straw. CONCLUSIONS: This is the first report describing the enrichment of lignocellulose degrading bacteria from the gut of the rice yellow stem borer to deconstruct rice straw, identifying a plethora of enzymes secreted by the microbial community when growing on rice straw as a carbon source. These enzymes could be important candidates for biorefineries to overcome the current bottlenecks in biomass processing.

Genome analysis of Paenibacillus polymyxa A18 gives insights into the features associated with its adaptation to the termite gut environment.

Paenibacillus polymyxa A18 was isolated from termite gut and was identified as a potential cellulase and hemicellulase producer in our previous study. Considering that members belonging to genus Paenibacillus are mostly free-living in soil, we investigated here the essential genetic features that helped P. polymyxa A18 to survive in gut environment. Genome sequencing and analysis identified 4608 coding sequences along with several elements of horizontal gene transfer, insertion sequences, transposases and integrated phages, which add to its genetic diversity. Many genes coding for carbohydrate-active enzymes, including the enzymes responsible for woody biomass hydrolysis in termite gut, were identified in P. polymyxa A18 genome. Further, a series of proteins conferring resistance to 11 antibiotics and responsible for production of 4 antibiotics were also found to be encoded, indicating selective advantage for growth and colonization in the gut environment. To further identify genomic regions unique to this strain, a BLAST-based comparative analysis with the sequenced genomes of 47 members belonging to genus Paenibacillus was carried out. Unique regions coding for nucleic acid modifying enzymes like CRISPR/Cas and Type I Restriction-Modification enzymes were identified in P. polymyxa A18 genome suggesting the presence of defense mechanism to combat viral infections in the gut. In addition, genes responsible for the formation of biofilms, such as Type IV pili and adhesins, which might be assisting P. polymyxa A18 in colonizing the gut were also identified in its genome. In situ colonization experiment further confirmed the ability of P. polymyxa A18 to colonize the gut of termite.

Disruption of zinc finger DNA binding domain in catabolite repressor Mig1 increases growth rate, hyphal branching, and cellulase expression in hypercellulolytic fungus Penicillium funiculosum NCIM1228.

BACKGROUND: There is an urgent requirement for second-generation bio-based industries for economical yet efficient enzymatic cocktail to convert diverse cellulosic biomass into fermentable sugars. In our previous study, secretome of Penicillium funiculosum NCIM1228 showed high commercial potential by exhibiting high biomass hydrolyzing efficiency. To develop NCIM1228 further as an industrial workhorse, one of the major genetic interventions needed is global deregulation of cellulolytic genes to achieve higher enzyme production. Mig1 orthologs found in all yeast and filamentous fungi are transcriptional regulators that maintain carbon homeostasis by negatively regulating genes of secondary carbon source utilization. Their disruption has long been known to be beneficial for increasing the production of secreted enzymes for alternate carbon source utilization. RESULTS: Upon detailed genotypic and phenotypic analysis, we observed that NCIM1228 harbors a truncated yet functional allele of homolog of a well-known catabolite repressor, Mig1. Alleviation of carbon repression in NCIM1228 was attained by replacing functional Mig1134 allele with null allele Mig188. P. funiculosum having Mig188 null allele showed better growth characteristics and 1.75-fold better glucose utilization than parent strain. We also showed that visibly small colony size, one of the major characteristics of CCR disruptant strains in filamentous fungi, was not due to retarded growth, but altered hyphal morphology. CCR-disrupted strain PfMig188 showed profuse branching pattern in terminal hyphae resulting in small and compact colonies with compromised filamentous proliferation. We further observed that basal level expression of two major classes of cellulases, namely, cellobiohydrolase and endoglucanase, was regulated by Mig1134 in NCIM1228, whereas other two major classes, namely, xylanases and ß-glucosidase, were only marginally regulated. Finally, CCR disruption in P. funiculosum NCIM1228 led to prolonged cellulase induction in production medium resulting in twofold increased cellulase activity than the parent strain with maximum secreted protein titer being > 14 g/l. CONCLUSIONS: CCR-disrupted P. funiculosum showed better growth, enhanced carbon source utilization, profuse branching pattern in terminal hyphae, and higher cellulase activity than parent strain. Our findings are particularly important in shedding light on important functions performed by Mig1 in addition to its role as negative regulator of alternate carbon source utilization in filamentous fungi.