Biotechnology for Biofuels

Lytic polysaccharide monooxygenases (LPMOs) are crucial industrial enzymes required in the biorefinery industry as well as in natural carbon cycle. These enzymes known to possess auxiliary activity are produced by numerous bacterial and fungal species to assist in the degradation of cellulosic biomass. In this study, we annotated and performed structural analysis of an uncharacterized thermostable LPMO from Penicillium funiculosum (PfLPMO9) in an attempt to understand nature of this enzyme in biomass degradation. PfLPMO9 exhibited 75% and 36% structural identity to Thermoascus aurantiacus (TaLPMO9A) and Lentinus similis (LsLPMO9A), respectively. Analysis of the molecular interactions during substrate binding revealed that PfLPMO9 demonstrated a higher binding affinity with a {Delta}G free energy of -46 k kcal/mol when compared with that of TaLPMO9A (-31 kcal/mol). The enzyme was further found to be highly thermostable at elevated temperature with a half-life of [~]88 h at 50 {degrees}C. Furthermore, multiple fungal genetic manipulation tools were employed to simultaneously overexpress this LPMO and Cellobiohydrolase I (CBH1) in catabolite derepressed strain of Penicillium funiculosum, PfMig188, in order to improve its saccharification performance towards acid pretreated wheat straw (PWS) at 20% substrate loading. The resulting transformants showed [~]200% and [~]66% increase in LPMO and Avicelase activities, respectively. While the secretomes of individually overexpressed LPMO and CBH1-strains increased saccharification of PWS by 6% and 13%, respectively, over PfMig188 at same enzyme concentration, the simultaneous overexpression of these two genes led to 20% increase in saccharification efficiency over PfMig188, which accounted for 82% saccharification of PWS at 20% substrate loading. ImportanceEnzymatic hydrolysis of cellulosic biomass by cellulases continues to be a significant bottleneck in the development of second-generation bio-based industries. While efforts are being intensified at how best to obtain indigenous cellulase for biomass hydrolysis, the high production cost of this enzyme remains a crucial challenge confronting its wide availability for efficient utilization of cellulosic materials. This is because it is challenging to get an enzymatic cocktail with balanced activity from a single host. This report provides for the first time the annotation and structural analysis of an uncharacterized thermostable lytic polysaccharide monooxygenase (LPMO) gene in Penicillium funiculosum and its impact in biomass deconstruction upon overexpression in catabolite derepressed strain of P. funiculosum. Cellobiohydrolase I (CBH1) which is the most important enzyme produced by many cellulolytic fungi for saccharification of crystalline cellulose was further overexpressed simultaneously with the LPMO. The resulting secretome was analyzed for enhanced LPMO and exocellulase activities with the corresponding improvement in its saccharification performance at high substrate loading by [~]20% using a minimal amount of protein.

BackgroundCellulose degradation by cellulases has been studied for decades due to the potential of using lignocellulosic biomass as a sustainable source of bioethanol. In plant cell walls, cellulose is bonded together and strengthened by the polyphenolic polymer, lignin. Because lignin is tightly linked to cellulose and is not digestible by cellulases, is thought to play a dominant role in limiting the efficient enzymatic degradation of plant biomass. Removal of lignin via pretreatments currently limits the cost-efficient production of ethanol from cellulose, motivating the need for a better understanding of how lignin inhibits cellulase-catalyzed degradation of lignocellulose. Work to date using bulk assays has suggested three possible inhibition mechanisms: lignin blocks access of the enzyme to cellulose, lignin impedes progress of the enzyme along cellulose, or lignin binds cellulases directly and acts as a sink. ResultsWe used single-molecule fluorescence microscopy to investigate the nanoscale dynamics of Cel7A from Trichoderma reesei, as it binds to and moves along purified bacterial cellulose in vitro. Lignified cellulose was generated by polymerizing coniferyl alcohol onto purified bacterial cellulose, and the degree of lignin incorporation into the cellulose meshwork was analyzed by optical and electron microscopy. We found that Cel7A preferentially bound to regions of cellulose where lignin was absent, and that in regions of high lignin density, Cel7A binding was inhibited. With increasing degrees of lignification, there was a decrease in the fraction of Cel7A that moved along cellulose rather than statically binding. Furthermore, with increasing lignification, the velocity of processive Cel7A movement decreased, as did the distance that individual Cel7A molecules moved during processive runs. ConclusionsIn an in vitro system that mimics lignified cellulose in plant cell walls, lignin did not act as a sink to sequester Cel7A and prevent it from interacting with cellulose. Instead, lignin both blocked access of Cel7A to cellulose and impeded the processive movement of Cel7A along cellulose. This work implies that strategies for improving biofuel production efficiency should target weakening interactions between lignin and cellulose surface, and further suggest that nonspecific adsorption of Cel7A to lignin is likely not a dominant mechanism of inhibition.

The oleaginous yeast Rhodotorula toruloides is a promising host for sustainable bioproduction due to its capacity to naturally utilize xylose present in lignocellulosic biomass, an abundant and renewable resource. However, its xylose consumption pathway is still not completely understood. To better understand the potential limitations in xylose utilization in R. toruloides, heterologous xylose reductase from Scheffersomyces stipitis, together with the native and heterologous xylulokinases from three different microorganisms (Scheffersomyces stipitis, Candida intermedia, and Escherichia coli) were overexpressed solely and in combination. The overexpression of xylulokinases showed more significant improvements in terms of xylose consumption rate compared to the single overexpression of xylose reductase. When the heterologous xylulokinase from Escherichia coli was overexpressed, the specific xylose consumption rate was improved by 66% and the maximum specific growth rate by 30% compared to the parental strain. The xylose specific consumption rate increased by 146% and the maximum specific growth rate increased by 118% when heterologous genes for xylose reductase and xylulokinase from E. coli were overexpressed together. These results suggest that the low expression of xylulokinase in R. toruloides, which has been reported previously, could limit its sugar consumption, while supporting higher lipid accumulation in this yeast.

Enzymatic lignocellulosic biomass conversion to bioethanol is dependent on efficient enzyme systems with {beta}-glucosidase as a key component. In this study, we performed in-depth profiling of the various {beta}-glucosidases present in the genome of the hypercellulolytic fungus; Penicillium funiculosum using genomics, transcriptomics, proteomics and molecular dynamics simulation approaches. Of the eight {beta}-glucosidase genes identified in the P. funiculosum genome, three were found to be extracellular, as evidenced by presence of signal peptides and mass spectrometry. Among the three secreted {beta}-glucosidase, two belonged to the GH3 and one belonged to GH1 families. Modelled structures of these proteins predicted a deep and narrow active site for the GH3 {beta}-glucosidases (PfBgl3A and PfBgl3B) and a shallow open active site for the GH1 {beta}-glucosidase (PfBgl1A). The enzymatic assays indicated that P. funiculosum secretome showed high {beta}-glucosidase activities with prominent bands on 4-methylumbelliferyl {beta}-D-glucopyranoside (MUG) zymogram. To understand the contributory effect of each of the three secreted {beta}-glucosidases (PfBgls), the corresponding gene was deleted separately and the effect of the deletion on {beta}-glucosidase activity of the secretome was examined. Although not the most abundant {beta}-glucosidase, PfBgl3A was found to be the most significant one as evidenced by a 42 % reduction in {beta}-glucosidase activity in the {Delta}PfBgl3A strain. To improve the thermostability, two mutants of PfBgl3A were designed with the help of molecular dynamics (MD) simulation and were expressed in Pichia pastoris for evaluation. The PfBgl3A mutant (Mutant A) gave 1.4 fold increase in the half-life (T1/2) of the enzyme at 50{degrees}C. IMPORTANCECommercially available cellulases are majorly produced from Trichoderma reesei. However, external supplementation of the cellulase cocktail from this host with exogenous {beta}-glucosidase is often required to achieve desired optimal saccharification of cellulosic feedstocks. This challenge has led to exploration of other cellulase-producing strains because of the importance of this class of enzymes in the cellulose deconstruction machinery. The non-model hypercellulolytic fungus Penicillium funiculosum has been studied in recent times and identified as a promising source of industrial cellulases. Various genetic interventions targeted at strain improvement for cellulase production have been performed. However, the {beta}-glucosidases of this strain have remained largely understudied. This study, therefore, reports profiling of all the eight {beta}-glucosidases of P. funiculosum via molecular and computational approaches and enhancing thermostability of the most promising {beta}-glucosidase via protein engineering. The results of this study set the background for future engineering strategies to transform the fungus into an industrial workhorse.

The primary challenge in utilizing palm kernel meal (PKM, an agricultural by-product) as non- ruminant livestock feed is its high fibre content, predominantly in the form of mannan. Microbial fermentation offers an economically favourable alternative to enzyme supplementation for breaking down fibre in lignocellulosic biomass. In a recent study, we have isolated and characterized an undomesticated strain (Bacillus subtilis F6) that is able to secrete mannanase. In this work, the mannanase production was substantially improved by optimizing multiple regulatory elements controlling the mannanase expression. Mannanase GmuG, sourced from B. subtilis F6 and verified for its hydrolytic activity on PKM fibre, was expressed using a replicative plasmid (pBE-S). The recombinant strain of B. subtilis F6 exhibited 1.9-fold increase in the mannanase activity during solid-state fermentation. Optimization of signal peptide and ribosome binding site further enhanced mannanase activity by 3.1-fold. Subsequently, promoter screening based on highly transcribed genes in B. subtilis F6 resulted in a significant 5.4-fold improvement in mannanase activity under the nprE promoter. The nprE promoter was further refined by eliminating specific transcription factor binding sites, enhancing the mannanase activity further by 1.8-fold. Notably, a substantial 35-40% reduction in PKM fibre content was observed after 30 h of fermentation using the recombinant strains. Lastly, the highest mannanase-producing strain was examined for scaled-up fermentation. The impacts of fermentation on fibre and protein contents, as well as the surface morphology of PKM, were analysed. The outcomes of this study offer an efficient method for robust mannanase expression in B. subtilis and its potential application in the biotransformation of PKM and other mannan-rich bioresources for improved feed utilization. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=83 SRC="FIGDIR/small/602432v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@10fbb9corg.highwire.dtl.DTLVardef@1e619fborg.highwire.dtl.DTLVardef@1b3bc0corg.highwire.dtl.DTLVardef@fec816_HPS_FORMAT_FIGEXP M_FIG C_FIG

AO_SCPLOWBSTRACTC_SCPLOWSimultaneous intracellular depolymerization of xylo-oligosaccharides (XOS) and acetate fermentation by engineered Saccharomyces cerevisiae offers an advance towards more cost-effective second-generation (2G) ethanol production. As xylan is one of the most abundant polysaccharides present in lignocellulosic residues, the transport and breakdown of XOS in an intracellular environment might bring a competitive advantage for recombinant strains in competition with contaminating microbes, which are always present in fermentation tanks; furthermore, acetic acid is a ubiquitous toxic component in lignocellulosic hydrolysates, deriving from hemicellulose and lignin breakdown. In the present work, the previously engineered S. cerevisiae strain, SR8A6S3, expressing NADPH-linked xylose reductase (XR), NAD+-linked xylitol dehydrogenase (XDH) (for xylose assimilation), as well as NADH-linked acetylating acetaldehyde dehydrogenase (AADH) and acetyl-CoA synthetase (ACS) (for an NADH-dependent acetate reduction pathway), was used as the host for expressing of two {beta}-xylosidases, GH43-2 and GH43-7, and a xylodextrin transporter, CDT-2, from Neurospora crassa, yielding the engineered strain SR8A6S3-CDT2-GH432/7. Both {beta}-xylosidases and the transporter were introduced by replacing two endogenous genes, GRE3 and SOR1, that encode aldose reductase and sorbitol (xylitol) dehydrogenase, respectively, which catalyse steps in xylitol production. Xylitol accumulation during xylose fermentation is a problem for 2G ethanol production since it reduces final ethanol yield. The engineered strain, SR8A6S3-CDT2-GH432/7, produced ethanol through simultaneous co-utilization of XOS, xylose, and acetate. The mutant strain produced 60% more ethanol and 12% less xylitol than the control strain when a hemicellulosic hydrolysate was used as a mono- and oligosaccharide source. Similarly, the ethanol yield was 84% higher for the engineered strain using hydrolysed xylan compared with the parental strain. The consumption of XOS, xylose, and acetate expands the capabilities of S. cerevisiae for utilization of all of the carbohydrate in lignocellulose, potentially increasing the efficiency of 2G biofuel production. HighlightsO_LIIntegration of XOS pathway in an acetate-xylose-consuming S. cerevisiae strain; C_LIO_LIIntracellular fermentation of XOS, acetate and xylose improved ethanol production; C_LIO_LIDeletion of both sor1{Delta} and gre3{Delta} reduced xylitol production. C_LI

Cellulases are a group of enzymes with several applications in biofuel production, and the paper, food, pharmaceutical, and chemical industries. Trichoderma harzianum P49P11 secrete all cellulases with high efficiency, representing an alternative to the current filamentous fungi in biotechnological industries. In this study, the cellulolytic mechanisms employed by the strain P49P11 to degrade crystalline cellulose in batch fermentation culture mode were elucidated by combining genome and secretome analysis. The strain P49P11 encodes nineteen cellulase genes from five different CAZyme families (GH5, GH6, GH7, GH12, and GH45), followed by several enzyme families for hemicellulose, pectin, and alpha-and beta-glucans degradation. The diverse CAZymes were also observed in the secretome, including cellulases, hemicellulases, and glucanases. In addition, {beta}-glucosidases and xylanase activities detected during the fermentation process validated our secretome analysis. Taken together, our results revealed all enzymatic machinery used by the T. harzianum P49P11 to degrade cellulose in batch fermentation mode. HighlightsO_LIWe described a high-quality genome assembly and annotation of the T. harzianum P49P11. C_LIO_LIThe T. harzianum P49P11 genome possesses a complete set of genes for lignocellulose degradation. C_LIO_LIThe first report on T. harzianum P49P11 secretome obtained from batch fermentation strategy. C_LIO_LIT. harzianum P49P11 produced cellulases, lignocellulases, and auxiliary enzymes produced in response to crystalline cellulose. C_LI

Ethanol production from renewable cellulosic materials is a globally significant research area. However, the high temperatures and acetic acid generated during cellulose pretreatment can inhibit Saccharomyces cerevisiae growth, reducing ethanol yields. This study investigates the impact of glutaredoxin family genes (GRXs) over-expression on S. cerevisiae cell growth and fermentation performance under thermal and acetic acid stress. Engineered strains overexpressing GRX1, GRX2, and GRX5 demonstrated enhanced growth at 42{degrees}C, while those overexpressing GRX1, GRX2, GRX6, and GRX7 showed improved growth at 1 g/L acetic acid. These results suggest that GRX over-expression can remediate S. cerevisiae, potentially accelerating advancements in green biomanufacturing.

The pectin lyase activity of 59 Bacillus amyloliquefaciens subsp. plantarum (Bap) strains was tested in vitro on Pectate Agar (PA) and Tris-Spizizen Salts (TSS) medium. Bap strains were cultured on TSA medium and washed three times with sterile water before the inoculation on PA media. Higher and lower pectate lyase activity were observed in six (AP193, AP203, AP299, AP80, AP102, and AP52) and four (AP 194, AP214, AP215, and AP305) Bap strains compared to other Bap strains. A total of 12 Bap strains (AP67, AP71, AP77, AP78, AP85, AP102, AP108, AP135, AP143, AP189, AP192, and AP193) grew vigorously on TSS medium. A total of six Bap strains (AP194, AP204, AP214, AP216, AP219, and HD73) had lower growth compared to other Bap strains. Pectin (1%) were used for in vitro PA and TSS medium. Pectate lyase and utilization activity were not found in Bacillus thuringiensis subsp. kurstaki strain HD73 compared to Bap strains. A draft genome sequence for strains AP194 and AP214 that were negative for pectin utilization were generated using an Illumina MiSeq. RAST analysis revealed that the pectin-associated gene altronate hydrolase (uxaA) absent in AP 214 strain. Multiple amino acid alignments of exuT and uxuB gene sequence showed dissimilarities among AP194, AP214, and reference Bap strains.

Lignocellulose biomass is one of the most abundant resources for sustainable biofuels. However, scaling up the biomass-to-biofuels conversion process for widespread usage is still pending. Bottlenecks during the process of enzymatic hydrolysis are the high cost of enzymes and the labor-intensive need for substrate-dependent enzyme mixtures. Current research efforts are therefore targeted at searching for or engineering lignocellulolytic enzymes of high efficiency. One way is to engineer multi-enzyme complexes that mimic the bacterial cellulosomal system, known to increase degradation efficiency up to 50-fold when compared to freely-secreted enzymes. However, these designer cellulosomes are instable and less efficient than wild type cellulosomes. Fungi cellulosomes discovered in recent years have significant differences from bacterial counterparts and hold great potential for industrial applications, both as designer cellulosomes and as additions to the enzymatic repertoire. Up to date, they are only found in a few anaerobic fungi. In this review, we extensively compared the degradation mechanisms in bacteria and fungi, and highlighted the essential gaps in applying these mechanisms in industrial applications. To better understand cellulosomes in microorganisms, we examined their sequences in 66,252 bacterial species and 823 fungal species and identified several bacterial species that are potentially cellulosome-producing. These findings act as a valuable resource in the biomass community for further proteomic and genetic sequence analysis. We also collated the current strategies of bioengineering lignocellulose degradation to suggest concepts that could be favorable for industrial usage.

In this study, we employ a two-stage dynamic metabolic control strategy to enhance the NADPH dependent biosynthesis of ethylene glycol from xylose in engineered E. coli. We evaluated the use of metabolic valves to dynamically reduce the enzymes involved in competitive pathways which compete for substrates with ethylene glycol biosynthesis, as well as regulatory pathways aimed at increasing NADPH fluxes. The performance of our initial strains with limits in pathway expression levels was improved by the addition of competitive valves, but not by increases in NADPH flux. In contrast, improving pathway expression levels, led to strains improved significantly by our regulatory valves which improved NADPH flux, but not by the competitive valves. This is consistent with a central hypothesis that faster pathways in and of themselves can compete with other metabolic fluxes by being faster and are better aided by regulatory changes capable of change rates elsewhere in metabolism. In this case in NADPH flux. Lastly, upon scale up to fed-batch bioreactors, our optimized strain, featuring dynamic control of two regulatory valves produced 140 g/L of EG in 70 hours at 92% of the theoretical yield.

A plethora of studies have focused on improvements of xylitol production. The challenges of establishing a biotechnological route for the industrial production of this sugar have been explored using different microorganisms and renewable feedstock. Nevertheless, sugarcane biomass has been neglected as the pentose source for xylitol production using Saccharomyces cerevisiae. Therefore, here we investigate the use of an industrial S. cerevisiae strain for xylitol production in batch fermentation of non-detoxified sugarcane straw hydrolysate, envisioning the diversification of the current infrastructure used for second-generation bioethanol production from the same lignocellulosic material. In order to optimize the xylose conversion in a non-fed cultivation system, guidelines in cell inoculum and medium supplementation are suggested, as well as the first attempt to use electro-fermentation for this purpose. Accordingly, our results show that the increase in initial cell density and hydrolysate supplementation allows a xylitol production of 19.24 {+/-} 0.68 g/L, representing 0,132 g/L.h productivity.

Clostridium thermocellum is a cellulolytic thermophile considered for consolidated bioprocessing of lignocellulose to ethanol. Improvements in ethanol yield are required for industrial implementation, but incompletely understood causes of amino acid secretion impede progress. In this study, amino acid secretion was investigated by gene deletions in ammonium-regulated NADPH-supplying and -consuming pathways and physiological characterization in cellobiose- or ammonium-limited chemostats. First, the contribution of the NADPH-supplying malate shunt was studied with strains using either the NADPH-yielding malate shunt ({Delta}ppdk) or redox-independent conversion of PEP to pyruvate ({Delta}ppdk {Delta}malE::Peno-pyk). In the latter, branched-chain amino acids, especially valine, were significantly reduced, whereas the ethanol yield increased 46-60%, suggesting that secretion of these amino acids balances NADPH surplus from the malate shunt. Unchanged amino acid secretion in {Delta}ppdk falsified a previous hypothesis on ammonium-regulated PEP-to-pyruvate flux redistribution. Possible involvement of another NADPH-supplier, namely NADH-dependent reduced ferredoxin:NADP+ oxidoreductase (nfnAB), was also excluded. Finally, deletion of glutamate synthase (gogat) in ammonium assimilation resulted in upregulation of NADPH-linked glutamate dehydrogenase activity and decreased amino acid yields. Since gogat in C. thermocellum is putatively annotated as ferredoxin-linked, which is supported by product redistribution observed in this study, this deletion likely replaced ferredoxin with NADPH in ammonium assimilation. Overall, these findings indicate that a need to reoxidize NADPH is driving the observed amino acid secretion, likely at the expense of NADH needed for ethanol formation. This suggests that metabolic engineering strategies on simplifying redox metabolism and ammonium assimilation can contribute to increased ethanol yields. ImportanceImproving the ethanol yield of C. thermocellum is important for industrial implementation of this microorganism in consolidated bioprocessing. A central role of NADPH in driving amino acid byproduct formation was demonstrated, by eliminating the NADPH-supplying malate shunt and separately by changing the cofactor specificity in ammonium assimilation. With amino acid secretion diverting carbon and electrons away from ethanol, these insights are important for further metabolic engineering to reach industrial requirements on ethanol yield. This study also provides chemostat data relevant for training genome-scale metabolic models and improving the validity of their predictions, especially considering the reduced degree-of-freedom in redox metabolism of the strains generated here. In addition, this study advances fundamental understanding on mechanisms underlying amino acid secretion in cellulolytic Clostridia as well as regulation and cofactor specificity in ammonium assimilation. Together, these efforts aid development of C. thermocellum for sustainable consolidated bioprocessing of lignocellulose to ethanol with minimal pretreatment.

Microbial lipid production from second generation feedstocks presents a sustainable route to future fuels, foods and bulk chemicals. The oleaginous yeast Metshnikowia pulcherrima has previously been investigated as a potential platform organism for lipid production due to its ability to be grown in non-sterile conditions and metabolising a wide range of oligo- and monosaccharide carbon sources within lignocellulosic hydrolysates. However, the generation of inhibitors from depolymerisation causes downstream bioprocessing complications, and despite M. pulcherrimas comparative tolerance, their presence is deleterious to both biomass and lipid formation. Using either a single inhibitor (formic acid) or an inhibitor cocktail (formic acid, acetic acid, fufural and HMF), two strategies of adaptive laboratory evolution were performed to improve M. pulcherrimas fermentation inhibitor tolerance. Using a sequential batch culturing approach, the resulting strains from both strategies had increased growth rates and reduced lag times under inhibiting conditions versus the progenitor. Interestingly, the lipid production of the inhibitor cocktail evolved strains markedly increased, with one strain producing 41% lipid by dry weight compared to 22% of the progenitor. The evolved species was cultured in a non-sterile 2L stirred tank bioreactor and accumulated lipid rapidly, yielding 6.1 g/L of lipid (35% cell dry weight) within 48 hours; a lipid productivity of 0.128 g L-1 h-1. Furthermore, the lipid profile was analogous to palm oil, consisting of 39% C16:0 and 56% C18:1 after 48 hours.

Lignocellulosic biomass (LCB) captures a major fraction of agro-industrial wastes that are mostly valorised for the production of second-generation biofuels. The extensive pre-treatment followed by saccharification of LCB restricts its usability for the production of a wide array of bioproducts. This study highlights the performance comparison of sono-assisted alkaline pre-treatment versus microbial pre-treatment of sugarcane bagasse by a no. of analytical techniques such as FTIR, XRD, CHNS, and SEM. Moreover, simultaneous delignification, saccharification and fermentation (SDSF) in one pot is highly desirable for the cost effective and environment friendly production of microbial products. In the present study SDSF was carried out by a cellulolytic bacterium Cellulomonas flavigena, for the production of extracellular polymeric substance (EPS), which is, by chemical nature a biopolymer made up of carbohydrate and protein subunits. The biopolymer was found to have an overall positive charge as analysed by ion exchange chromatography. Two major fractions of molecular weight 237 KDa and 29 KDa were obtained from gel filtration chromatography, in addition to that, the EPS was found to be composed of monosaccharides, D (+) mannosamine and D (+) xylose as identified from high pressure liquid chromatography (HPLC).

Efficient, large-scale heterologous production of enzymes is a crucial component of the biomass valorization industry. Whereas cellulose utilization has been successful in applications such as bioethanol, its counterpart lignin remains significantly underutilized despite being an abundant potential source of aromatic commodity chemicals. Fungal lignin-degrading heme peroxidases are thought to be the major agents responsible for lignin depolymerization in nature, but their large-scale production remains inaccessible due to the genetic intractability of basidiomycete fungi and the challenges in the heterologous production of these enzymes. In this study, we employ a strain engineering approach based on functional genomics to identify mutants of the model yeast Saccharomyces cerevisiae with enhanced heterologous production of lignin-degrading heme peroxidases. We show that our screening method coupling an activity-based readout with fluorescence-assisted cell sorting enables identification of two single null mutants of S. cerevisiae, pmt2 and cyt2, with up to 11-fold improved secretion of a versatile peroxidase from the lignin-degrading fungus Pleurotus eryngii. We demonstrate that the double deletion strain pmt2cyt2 displays positive epistasis, improving and even enabling production of members from all three classes of lignin-degrading fungal peroxidases. We anticipate that these mutant strains will be broadly applicable for improved heterologous production of this biotechnologically important class of enzymes.

Recent research endeavors have turned to sustainably generating useful chemicals from biological platforms. However, conventional model organisms, such as Escherichia coli and Saccharomyces cerevisiae, face limitations, particularly in terms of substrate range and yield for certain metabolites. In this study, we share our work toward the development of the non-model bacterium, Paraburkholderia sacchari (hereafter P. sacchari), as a microbial factory for the production of polyhydroxyalkanoates (PHAs), which are precursors for biodegradable plastic. The particular PHAs of interest produced by P. sacchari include poly(3-hydroxybutyrate) (PHB) and the co-polymer produced by the combination of PHB and 3-hydroxyvalerate (3HV) called poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). P. sacchari produces PHB from mixtures of hexose and pentose sugars commonly found in lignocellulosic biomass, however PHBV requires co-feeding with propionate. Both plastic precursors have industrial interest, so both PHB and 3HV were chosen as production targets. Due to studies in other bacteria demonstrating PHB yield can be improved by overexpressing genes for critical pathway enzymes, we hypothesized there is a bottleneck in the production pathway leading to PHB in P. sacchari as well. To explore this, heterologous genes coding for the three critical enzymes were taken from Cupriavidus necator H16 (hereafter C. necator) and inserted via plasmid; phaA and bktb (homologous genes for {beta}-ketothiolase), phaB (acetyl-CoA reductase), and phaC (PHA polymerase). PHB production increased following overexpression of phaB, indicating acetoacetyl-CoA as the limiting enzyme. In fact, overexpression of phaB with the synthetic Anderson promoter, BBa_J23 104, increased titer by 162% over wildtype. On the other hand, strategies to improve 3HV had mixed results. Heterologous overexpression of propionyl-CoA transferase (pct from C. necator), which converts propionate into propionyl-CoA-the starting substrate for the 3HVproduction, showed a 145% increase in 3HV. Yet, internal sourcing of propionyl-CoA from succinyl-CoA following introduction of the sleeping beauty mutase (sbm) operon from E. coli showed no 3HV production. To this end, Max/Min Driving Force (MDF) thermodynamic analysis of critical PHBV pathways revealed two major limitations of 3HV production: 1) internal sourcing is not thermodynamically favorable; and 2) recycling of propionyl-CoA through the methyl citrate cycle (MCC) is more favorable than 3HV formation. Overall, we have shown promising progress and suggest future directions toward an industrially useful strain of P. sacchari for PHB and PHBV production. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=71 SRC="FIGDIR/small/624694v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@1942ebaorg.highwire.dtl.DTLVardef@187ec83org.highwire.dtl.DTLVardef@b8b439org.highwire.dtl.DTLVardef@4015d6_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsParaburkholderia sacchari has interest as a bioproduction platform for PHAs from complex feedstocks. Removal of PHA pathway bottleneck increases PHB yield by 162%. Improved conversion of fed-in propionate increases 3HV yield by 145%. Internal sourcing of propionyl-CoA does not successfully yield 3HV. Thermodynamic analysis provides insight into difficult conversion of propionyl-CoA to 3HV.

Pseudomonas putida is a promising bacterial chassis for metabolic engineering given its ability to metabolize a wide array of carbon sources, especially aromatic compounds derived from lignin. However, this omnivorous metabolism can also be a hindrance when it can naturally metabolize products produced from engineered pathways. Herein we show that P. putida is able to use valerolactam as a sole carbon source, as well as degrade caprolactam. Lactams represent important nylon precursors, and are produced in quantities exceeding one million tons per year[1]. To better understand this metabolism we use a combination of Random Barcode Transposon Sequencing (RB-TnSeq) and shotgun proteomics to identify the oplBA locus as the likely responsible amide hydrolase that initiates valerolactam catabolism. Deletion of the oplBA genes prevented P. putida from growing on valerolactam, prevented the degradation of valerolactam in rich media, and dramatically reduced caprolactam degradation under the same conditions. Deletion of oplBA, as well as pathways that compete for precursors L-lysine or 5-aminovalerate, increased the titer of valerolactam from undetectable after 48 hours of production to ~90 mg/L. This work may serve as a template to rapidly eliminate undesirable metabolism in non-model hosts in future metabolic engineering efforts.

As one of the top value-added chemicals, succinic acid has been the focus of numerous metabolic engineering campaigns since the 1990s. However, microbial production of succinic acid at an industrially relevant scale has been hindered by high downstream processing costs arising from neutral pH fermentation. Here we describe the metabolic engineering of Issatchenkia orientalis, a non-conventional yeast with superior tolerance to highly acidic conditions, for cost-effective succinic acid production. Through deletion of byproduct pathways, transport engineering, and expanding the substrate scope, the resulting strains could produce succinic acid at the highest titers in sugar-based media at low pH (pH 3) in fed-batch fermentations using bench-top reactors, i.e. 109.5 g/L in minimal medium and 104.6 g/L in sugarcane juice medium. We further performed batch fermentation in a pilot-scale fermenter with a scaling factor of 300x, achieving 63.1 g/L of succinic acid using sugarcane juice medium. A downstream processing comprising of two-stage vacuum distillation and crystallization enabled direct recovery of succinic acid, without further acidification of fermentation broth, with an overall yield of 64.0%. Finally, we simulated an end-to-end low-pH succinic acid production pipeline, and techno-economic analysis and life cycle assessment indicate our process is financially viable and can reduce life cycle greenhouse gas emissions by 34-90% relative to fossil-based production processes. We expect I. orientalis can serve as a general industrial platform for the production of a wide variety of organic acids.

Ethanol, a naturally synthesized compound by Saccharomyces cerevisiae yeast through alcoholic fermentation, has previously been studied as a renewable alternative to traditional fossil fuels. However, current challenges of engineering S. cerevisiae strains for ethanol production remain: low ethanol productivity, inefficient substrate catabolism, and a buildup of toxic products to inhibitory levels. In this study, we proposed a method of metabolic rewiring via the deletion of the pda1 gene, which leads to pyruvate dehydrogenase (PDH) deficiency. The {Delta}pda1 mutant strain was created by CRISPR Cas-9 knockout using the constructed pCRCT-PDA1 plasmid. Subsequently, mutant candidates were screened by PCR and Sanger sequencing, confirming a 17 bp deletion in the pda1 gene. The wild-type and mutant strains were analyzed for growth under aerobic and anaerobic conditions in glucose and glycerol, as well as ethanol production and tolerance. The {Delta}pda1 mutant displays a ~two-fold increase in anaerobic ethanol production and an aerobic growth defect with no observed increase in ethanol production. The mutant is also hyper-tolerant to ethanol, which allows a faster buildup of products in growth media with minimal reduction in growth. This new S. cerevisiae strain deficient in PDH may provide a solution to the efficient and abundant synthesis of biofuels such as ethanol by redirecting metabolic flux and altering stress response.