Calcium signaling positively regulates cellulase translation and secretion in a Clr-2-overexpressing, catabolically derepressed strain of Penicillium funiculosum.

BACKGROUND: Low-cost cellulase production is vital to sustainable second-generation biorefineries. The catabolically derepressed strain of Penicillium funiculosum NCIM1228 (PfMig188 or ∆Mig1) secretes a superior set of cellulolytic enzymes, that are most suitable for 2G biorefineries. At a 3% (w/w) load, the ∆Mig1 secretome can release > 80% of fermentable sugars from lignocellulose at a 15% (w/v) biomass load, irrespective of the type of biomass and pretreatment. The robustness of the secretome can be further increased by improving the cellulase production capacity of the fungal strain. RESULTS: We began by identifying the transcription factor responsible for cellulase production in NCIM1228. An advanced RNA-seq screen identified three genes, clr-2, ctf1a and ctf1b; the genes were cloned under their native promoters and transformed into NCIM1228. Of the three, clr-2 overexpression led to twofold higher cellulase production than the parent strain and was thus identified as the transcriptional activator of cellulase in NCIM1228. Next, we overexpressed clr-2 in ∆Mig1 and expected an exponential increase in cellulolytic attributes accredited to the reinforced activation mechanisms, conjoint with diminished negative regulation. Although clr-2 overexpression increased the transcript levels of cellulase genes in ∆Mig1, there was no increase in cellulase yield. Even a further increase in the transcript levels of clr-2 via a stronger promoter was ineffective. However, when the CaCO3 concentration was increased to 5 g/l in the growth medium, we achieved a 1.5-fold higher activity of 6.4 FPU/ml in the ∆Mig1 strain with clr-2 overexpression. Enthused by the calcium effect, a transcriptomic screen for genes encoding Ca2+-activated kinase identified ssp1, whose overexpression could further increase cellulase yield to ~ 7.5 FPU/ml. Investigation of the mechanism revealed that calcium signaling exclusively enhances the translation and secretion of cellulase in Penicillium funiculosum. CONCLUSIONS: Our study identifies for the first time that cellulose activates two discrete signaling events to govern cellulase transcription and posttranscriptional processes (translation, processing and secretion) in P. funiculosum NCIM1228. Whereas Clr-2, the transcriptional activator of cellulase, governs transcription, calcium signaling specifically activates cellulase translation and secretion.

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.

Improved Butanol Production Using FASII Pathway in E. coli.

n-Butanol is often considered a potential substitute for gasoline due to its physicochemical properties being closely related to those of gasoline. In this study, we extend our earlier work to convert endogenously producing butyrate via the FASII pathway using thioesterase TesBT to its corresponding alcohol, i.e., butanol. We first assembled pathway genes, i.e., car encoding carboxylic acid reductase from Mycobacterium marinum, sfp encoding phosphopantetheinyl transferase from Bacillus subtilis, and adh2 encoding alcohol dehydrogenase from S. cerevisiae, responsible for bioconversion of butyrate to butanol in three different configurations (Operon, Pseudo-Operon, and Monocistronic) to achieve optimum expression of each gene and compared with the clostridial solventogenic pathway for in vivo conversion of butyrate to butanol under aerobic conditions. An E. coli strain harboring car, sfp, and adh2 in pseudo-operon configuration was able to convert butyrate to butanol with 100% bioconversion efficiency when supplemented with 1 g/L of butyrate. Further, co-cultivation of an upstream strain (butyrate-producing) with a downstream strain (butyrate to butanol converting) at different inoculation ratios was investigated, and an optimized ratio of 1:4 (upstream strain: downstream strain) was found to produce ∼2 g/L butanol under fed-batch fermentation. Further, a mono-cultivation approach was applied by transforming a plasmid harboring tesBT gene into the downstream strain. This approach produced 0.42 g/L in a test tube and ∼2.9 g/L butanol under fed-batch fermentation. This is the first report where both mono- and co-cultivation approaches were tested and compared for butanol production, and butanol titers achieved using both strategies are the highest reported values in recombinant E. coli utilizing FASII pathway.

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.

Advances in the development and application of microbial consortia for metabolic engineering.

Recent advances in metabolic engineering enable the production of high-value chemicals via expressing complex biosynthetic pathways in a single microbial host. However, many engineered strains suffer from poor product yields due to redox imbalance and excess metabolic burden, and require compartmentalization of the pathway for optimal function. To address this problem, significant developments have been made towards co-cultivation of more than one engineered microbial strains to distribute metabolic burden between the co-cultivation partners and improve the product yield. In this emerging approach, metabolic pathway modules can be optimized separately in suitable hosts that will then be combined to enable optimal functionality of the complete pathway. This modular approach broadens the possibilities to fine tune sophisticated production platforms and thus achieve the biosynthesis of very complex compounds. Here, we review the different applications and the overall potential of natural and artificial co-cultivation systems in metabolic engineering in order to improve bioproduction/bioconversion. In addition to the several advantages over monocultures, major challenges and opportunities associated with co-cultivation are also discussed in this review.

Complete Genome Sequence of Cupriavidus necator H16 (DSM 428).

The hydrogen-utilizing strain Cupriavidus necator H16 (DSM 428) was sequenced using a combination of PacBio and Illumina sequencing. Annotation of this strain reveals 6,543 protein-coding genes, 263 pseudogenes, 64 tRNA genes, and 15 rRNA genes.

CRISPR/Cas9-mediated engineering of Escherichia coli for n-butanol production from xylose in defined medium.

Butanol production from agricultural residues is the most promising alternative for fossil fuels. To reach the economic viability of biobutanol production, both glucose and xylose should be utilized and converted into butanol. Here, we engineered a dual-operon-based synthetic pathway in the genome of E. coli MG1655 to produce n-butanol using CRISPR/Cas9 technology. Further deletion of competing pathway followed by fed-batch cultivation of the engineered strain in a bioreactor with glucose-containing complex medium yielded 5.4 g/L n-butanol along with pyruvate as major co-product, indicating a redox imbalance. To ferment xylose into butanol in redox-balanced manner, we selected SSK42, an ethanologenic E. coli strain engineered and evolved in our laboratory to produce ethanol from xylose, for integrating synthetic butanol cassette in its genome via CRISPR/Cas9 after deleting the gene responsible for endogenous ethanol production. The engineered plasmid- and marker-free strain, ASA02, produced 4.32 g/L butanol in fed-batch fermentation in completely defined AM1-xylose medium.

Engineered Production of Short Chain Fatty Acid in Escherichia coli Using Fatty Acid Synthesis Pathway.

Short-chain fatty acids (SCFAs), such as butyric acid, have a broad range of applications in chemical and fuel industries. Worldwide demand of sustainable fuels and chemicals has encouraged researchers for microbial synthesis of SCFAs. In this study we compared three thioesterases, i.e., TesAT from Anaerococcus tetradius, TesBF from Bryantella formatexigens and TesBT from Bacteroides thetaiotaomicron, for production of SCFAs in Escherichia coli utilizing native fatty acid synthesis (FASII) pathway and modulated the genetic and bioprocess parameters to improve its yield and productivity. E. coli strain expressing tesBT gene yielded maximum butyric acid titer at 1.46 g L-1, followed by tesBF at 0.85 g L-1 and tesAT at 0.12 g L-1. The titer of butyric acid varied significantly depending upon the plasmid copy number and strain genotype. The modulation of genetic factors that are known to influence long chain fatty acid production, such as deletion of the fadD and fadE that initiates the fatty acid degradation cycle and overexpression of fadR that is a global transcriptional activator of fatty acid biosynthesis and repressor of degradation cycle, did not improve the butyric acid titer significantly. Use of chemical inhibitor cerulenin, which restricts the fatty acid elongation cycle, increased the butyric acid titer by 1.7-fold in case of TesBF, while it had adverse impact in case of TesBT. In vitro enzyme assay indicated that cerulenin also inhibited short chain specific thioesterase, though inhibitory concentration varied according to the type of thioesterase used. Further process optimization followed by fed-batch cultivation under phosphorous limited condition led to production of 14.3 g L-1 butyric acid and 17.5 g L-1 total free fatty acid at 28% of theoretical yield. This study expands our understanding of SCFAs production in E. coli through FASII pathway and highlights role of genetic and process optimization to enhance the desired product.

Identification of long chain specific aldehyde reductase and its use in enhanced fatty alcohol production in E. coli.

Long chain fatty alcohols have wide application in chemical industries and transportation sector. There is no direct natural reservoir for long chain fatty alcohol production, thus many groups explored metabolic engineering approaches for its microbial production. Escherichia coli has been the major microbial platform for this effort, however, terminal endogenous enzyme responsible for converting fatty aldehydes of chain length C14-C18 to corresponding fatty alcohols is still been elusive. Through our in silico analysis we selected 35 endogenous enzymes of E. coli having potential of converting long chain fatty aldehydes to fatty alcohols and studied their role under in vivo condition. We found that deletion of ybbO gene, which encodes NADP(+) dependent aldehyde reductase, led to >90% reduction in long chain fatty alcohol production. This feature was found to be strain transcending and reinstalling ybbO gene via plasmid retained the ability of mutant to produce long chain fatty alcohols. Enzyme kinetic study revealed that YbbO has wide substrate specificity ranging from C6 to C18 aldehyde, with maximum affinity and efficiency for C18 and C16 chain length aldehyde, respectively. Along with endogenous production of fatty aldehyde via optimized heterologous expression of cyanobaterial acyl-ACP reductase (AAR), YbbO overexpression resulted in 169mg/L of long chain fatty alcohols. Further engineering involving modulation of fatty acid as well as of phospholipid biosynthesis pathway improved fatty alcohol production by 60%. Finally, the engineered strain produced 1989mg/L of long chain fatty alcohol in bioreactor under fed-batch cultivation condition. Our study shows for the first time a predominant role of a single enzyme in production of long chain fatty alcohols from fatty aldehydes as well as of modulation of phospholipid pathway in increasing the fatty alcohol production.

A constitutive expression system for cellulase secretion in Escherichia coli and its use in bioethanol production.

The production of biofuels from lignocellulosic biomass appears to be attractive and viable due to the abundance and availability of this biomass. The hydrolysis of this biomass, however, is challenging because of the complex lignocellulosic structure. The ability to produce hydrolytic cellulase enzymes in a cost-effective manner will certainly accelerate the process of making lignocellulosic ethanol production a commercial reality. These cellulases may need to be produced aerobically to generate large amounts of protein in a short time or anaerobically to produce biofuels from cellulose via consolidated bioprocessing. Therefore, it is important to identify a promoter that can constitutively drive the expression of cellulases under both aerobic and anaerobic conditions without the need for an inducer. Using lacZ as reporter gene, we analyzed the strength of the promoters of four genes, namely lacZ, gapA, ldhA and pflB, and found that the gapA promoter yielded the maximum expression of the ß-galactosidase enzyme under both aerobic and anaerobic conditions. We further cloned the genes for two cellulolytic enzymes, ß-1,4-endoglucanase and ß-1,4-glucosidase, under the control of the gapA promoter, and we expressed these genes in Escherichia coli, which secreted the products into the extracellular medium. An ethanologenic E. colistrain transformed with the secretory ß-glucosidase gene construct fermented cellobiose in both defined and complex medium. This recombinant strain also fermented wheat straw hydrolysate containing glucose, xylose and cellobiose into ethanol with an 85% efficiency of biotransformation. An ethanologenic strain that constitutively secretes a cellulolytic enzyme is a promising platform for producing lignocellulosic ethanol.