Arabinose as an overlooked sugar for microbial bioproduction of chemical building blocks.

The circular economy is anticipated to bring a disruptive transformation in manufacturing technologies. Robust and industrial scalable microbial strains that can simultaneously assimilate and valorize multiple carbon substrates are highly desirable, as waste bioresources contain substantial amounts of renewable and fermentable carbon, which is diverse. Lignocellulosic biomass (LCB) is identified as an inexhaustible and alternative resource to reduce global dependence on oil. Glucose, xylose, and arabinose are the major monomeric sugars in LCB. However, primary research has focused on the use of glucose. On the other hand, the valorization of pentose sugars, xylose, and arabinose, has been mainly overlooked, despite possible assimilation by vast microbial communities. The present review highlights the research efforts that have explicitly proven the suitability of arabinose as the starting feedstock for producing various chemical building blocks via biological routes. It begins by analyzing the availability of various arabinose-rich biorenewable sources that can serve as potential feedstocks for biorefineries. The subsequent section outlines the current understanding of arabinose metabolism, biochemical routes prevalent in prokaryotic and eukaryotic systems, and possible products that can be derived from this sugar. Further, currently, exemplar products from arabinose, including arabitol, 2,3-butanediol, 1,2,3-butanetriol, ethanol, lactic acid, and xylitol are discussed, which have been produced by native and non-native microbial strains using metabolic engineering and genome editing tools. The final section deals with the challenges and obstacles associated with arabinose-based production, followed by concluding remarks and prospects.

Impact of the T296S mutation in P450 GcoA for aryl-O-demethylation: a QM/MM study.

Lignin, a complex plant cell wall component, holds promise as a renewable aromatic carbon feedstock. p-Vanillin is a key product of lignin depolymerization and a precursor of protocatechuic acid (PCA) that has tremendous potential for biofuel production. While the GcoAB enzyme, native to Amycolatopsis sp., naturally catalyzes aryl-O-demethylation toward guaiacol, recent research introduced a single mutation, T296S, into the GcoAP450 enzyme, enabling it to catalyze aryl-O-demethylation of p-vanillin. This structural modification increases the efficiency of GcoAP450 for the natural substrate while being active for p-vanillin. This study reveals the increased flexibility of p-vanillin and its ability to adapt a favorable conformation by aligning the methoxy group in close proximity to Fe(IV) = O of Cpd I in the active site of the T296S variant. The QM/MM calculations in accordance with the experimental data validated that the rate-limiting step for the oxidation of p-vanillin is hydrogen atom abstraction and provided a detailed geometric structure of stationary and saddle points for the oxidation of p-vanillin.

Metabolic engineering of Cupriavidus necator H16 for heterotrophic and autotrophic production of 3-hydroxypropionic acid.

3-Hydroxypropionate (3-HP) is a versatile compound for chemical synthesis and a potential building block for biodegradable polymers. Cupriavidus necator H16, a facultative chemolithoautotroph, is an attractive production chassis and has been extensively studied as a model organism for biopolymer production. Here, we engineered C. necator H16 for 3-HP biosynthesis from its central metabolism. Wild type C. necator H16 can use 3-HP as a carbon source, a highly undesirable trait for a 3-HP production chassis. However, deletion of its three (methyl-)malonate semialdehyde dehydrogenases (mmsA1, mmsA2 and mmsA3) resulted in a strain that cannot grow on 3-HP as the sole carbon source, and this strain was selected as our production host. A stepwise approach was used to construct pathways for 3-HP production via ß-alanine. Two additional gene deletion targets were identified during the pathway construction process. Deletion of the 3-hydroxypropionate dehydrogenase, encoded by hpdH, prevented the re-consumption of the 3-HP produced by our engineered strains, while deletion of gdhA1, annotated as a glutamate dehydrogenase, prevented the utilization of aspartate as a carbon source, one of the key pathway intermediates. The final strain carrying these deletions was able to produce up to 8 mM 3-HP heterotrophically. Furthermore, an engineered strain was able to produce 0.5 mM 3-HP under autotrophic conditions, using CO2 as sole carbon source. These results form the basis for establishing C. necator H16 as an efficient platform for the production of 3-HP and 3-HP-containing polymers.

Development of Hypertolerant Strain of Yarrowia lipolytica Accumulating Succinic Acid Using High Levels of Acetate.

Acetate is emerging as a promising feedstock for biorefineries as it can serve as an alternate carbon source for microbial cell factories. In this study, we expressed acetyl-CoA synthase in Yarrowia lipolytica PSA02004PP, and the recombinant strain grew on acetate as the sole carbon source and accumulated succinic acid or succinate (SA). Unlike traditional feedstocks, acetate is a toxic substrate for microorganisms; therefore, the recombinant strain was further subjected to adaptive laboratory evolution to alleviate toxicity and improve tolerance against acetate. At high acetate concentrations, the adapted strain Y. lipolytica ACS 5.0 grew rapidly and accumulated lipids and SA. Bioreactor cultivation of ACS 5.0 with 22.5 g/L acetate in a batch mode resulted in a maximum cell OD600 of 9.2, with lipid and SA accumulation being 0.84 and 5.1 g/L, respectively. However, its fed-batch cultivation yielded a cell OD600 of 23.5, SA titer of 6.5 g/L, and lipid production of 1.5 g/L with an acetate uptake rate of 0.2 g/L h, about 2.86 times higher than the parent strain. Cofermentation of acetate and glucose significantly enhanced the SA titer and lipid accumulation to 12.2 and 1.8 g/L, respectively, with marginal increment in cell growth (OD600: 26.7). Furthermore, metabolic flux analysis has drawn insights into utilizing acetate for the production of metabolites that are downstream to acetyl-CoA. To the best of our knowledge, this is the first report on SA production from acetate by Y. lipolytica and demonstrates a path for direct valorization of sugar-rich biomass hydrolysates with elevated acetate levels to SA.

Engineering Cupriavidus necator H16 for the autotrophic production of (R)-1,3-butanediol.

Butanediols are widely used in the synthesis of polymers, specialty chemicals and important chemical intermediates. Optically pure R-form of 1,3-butanediol (1,3-BDO) is required for the synthesis of several industrial compounds and as a key intermediate of ß-lactam antibiotic production. The (R)-1,3-BDO can only be produced by application of a biocatalytic process. Cupriavidus necator H16 is an established production host for biosynthesis of biodegradable polymer poly-3-hydroxybutryate (PHB) via acetyl-CoA intermediate. Therefore, the utilisation of acetyl-CoA or its upstream precursors offers a promising strategy for engineering biosynthesis of value-added products such as (R)-1,3-BDO in this bacterium. Notably, C. necator H16 is known for its natural capacity to fix carbon dioxide (CO2) using hydrogen as an electron donor. Here, we report engineering of this facultative lithoautotrophic bacterium for heterotrophic and autotrophic production of (R)-1,3-BDO. Implementation of (R)-3-hydroxybutyraldehyde-CoA- and pyruvate-dependent biosynthetic pathways in combination with abolishing PHB biosynthesis and reducing flux through the tricarboxylic acid cycle enabled to engineer strain, which produced 2.97 g/L of (R)-1,3-BDO and achieved production rate of nearly 0.4 Cmol Cmol-1 h-1 autotrophically. This is first report of (R)-1,3-BDO production from CO2.

Engineering improved ethylene production: Leveraging systems biology and adaptive laboratory evolution.

Ethylene is a small hydrocarbon gas widely used in the chemical industry. Annual worldwide production currently exceeds 150 million tons, producing considerable amounts of CO2 contributing to climate change. The need for a sustainable alternative is therefore imperative. Ethylene is natively produced by several different microorganisms, including Pseudomonas syringae pv. phaseolicola via a process catalyzed by the ethylene-forming enzyme (EFE), subsequent heterologous expression of EFE has led to ethylene production in non-native bacterial hosts including Escherichia coli and cyanobacteria. However, solubility of EFE and substrate availability remain rate-limiting steps in biological ethylene production. We employed a combination of genome-scale metabolic modelling, continuous fermentation, and protein evolution to enable the accelerated development of a high efficiency ethylene producing E. coli strain, yielding a 49-fold increase in production, the most significant improvement reported to date. Furthermore, we have clearly demonstrated that this increased yield resulted from metabolic adaptations that were uniquely linked to EFE (wild type versus mutant). Our findings provide a novel solution to deregulate metabolic bottlenecks in key pathways, which can be readily applied to address other engineering challenges.

A Sustainable Chemicals Manufacturing Paradigm Using CO2 and Renewable H2.

The chemical industry must decarbonize to align with UN Sustainable Development Goals. A shift toward circular economies makes CO2 an attractive feedstock for producing chemicals, provided renewable H2 is available through technologies such as supercritical water (scH2O) gasification. Furthermore, high carbon and energy efficiency is paramount to favorable techno-economics, which poses a challenge to chemo-catalysis. This study demonstrates continuous gas fermentation of CO2 and H2 by the cell factory, Cupriavidus necator, to (R,R)-2,3-butanediol and isopropanol as case studies. Although a high carbon efficiency of 0.75 [(C-mol product)/(C-mol CO2)] is exemplified, the poor energy efficiency of biological CO2 fixation requires ∼8 [(mol H2)/(mol CO2)], which is techno-economically infeasible for producing commodity chemicals. Heat integration between exothermic gas fermentation and endothermic scH2O gasification overcomes this energy inefficiency. This study unlocks the promise of sustainable manufacturing using renewable feedstocks by combining the carbon efficiency of bio-catalysis with energy efficiency enforced through process engineering.

Nanorg Microbial Factories: Light-Driven Renewable Biochemical Synthesis Using Quantum Dot-Bacteria Nanobiohybrids.

Living cells do not interface naturally with nanoscale materials, although such artificial organisms can have unprecedented multifunctional properties, like wireless activation of enzyme function using electromagnetic stimuli. Realizing such interfacing in a nanobiohybrid organism (or nanorg) requires (1) chemical coupling via affinity binding and self-assembly, (2) the energetic coupling between optoelectronic states of artificial materials with the cellular process, and (3) the design of appropriate interfaces ensuring biocompatibility. Here we show that seven different core-shell quantum dots (QDs), with excitations ranging from ultraviolet to near-infrared energies, couple with targeted enzyme sites in bacteria. When illuminated by light, these QDs drive the renewable production of different biofuels and chemicals using carbon-dioxide (CO2), water, and nitrogen (from air) as substrates. These QDs use their zinc-rich shell facets for affinity attachment to the proteins. Cysteine zwitterion ligands enable uptake through the cell, facilitating cell survival. Together, these nanorgs catalyze light-induced air-water-CO2 reduction with a high turnover number (TON) of ∼106-108 (mols of product per mol of cells) to biofuels like isopropanol (IPA), 2,3-butanediol (BDO), C11-C15 methyl ketones (MKs), and hydrogen (H2); and chemicals such as formic acid (FA), ammonia (NH3), ethylene (C2H4), and degradable bioplastics polyhydroxybutyrate (PHB). Therefore, these resting cells function as nanomicrobial factories powered by light.

Substrates and oxygen dependent citric acid production by Yarrowia lipolytica: insights through transcriptome and fluxome analyses.

BACKGROUND: Unlike the well-studied backer yeast where catabolite repression represents a burden for mixed substrate fermentation, Yarrowia lipolytica, an oleaginous yeast, is recognized for its potential to produce single cell oils and citric acid from different feedstocks. These versatilities of Y. lipolytica with regards to substrate utilization make it an attractive host for biorefinery application. However, to develop a commercial process for the production of citric acid by Y. lipolytica, it is necessary to better understand the primary metabolism and its regulation, especially for growth on mixed substrate. RESULTS: Controlling the dissolved oxygen concentration (pO2) in Y. lipolytica cultures enhanced citric acid production significantly in cultures grown on glucose in mono- or dual substrate fermentations, whereas with glycerol as mono-substrate no significant effect of pO2 was found on citrate production. Growth on mixed substrate with glucose and glycerol revealed a relative preference of glycerol utilization by Y. lipolytica. Under optimized conditions with pO2 control, the citric acid titer on glucose in mono- or in dual substrate cultures was 55 and 50 g/L (with productivity of 0.6 g/L*h in both cultures), respectively, compared to a maximum of 18 g/L (0.2 g/L*h) with glycerol in monosubstrate culture. Additionally, in dual substrate fermentation, glycerol limitation was found to trigger citrate consumption despite the presence of enough glucose in pO2-limited culture. The metabolic behavior of this yeast on different substrates was investigated at transcriptomic and 13C-based fluxomics levels. CONCLUSION: Upregulation of most of the genes of the pentose phosphate pathway was found in cultures with highest citrate production with glucose in mono- or in dual substrate fermentation with pO2 control. The activation of the glyoxylate cycle in the oxygen limited cultures and the imbalance caused by glycerol limitation might be the reason for the re-consumption of citrate in dual substrate fermentations. This study provides interesting targets for metabolic engineering of this industrial yeast.

Glucose-mediated regulation of glycerol uptake in Rhodosporidium toruloides: Insights through transcriptomic analysis on dual substrate fermentation.

Rhodosporidium toruloides is a robust oleaginous yeast that can accumulate lipids to more than 70% of its dry cell mass. Even though it is extensively studied for its fermentation of substrates like glucose and glycerol, limited information is available about its metabolism of mixture of glucose and glycerol. During growth on mixture of glucose and glycerol a typical diauxic growth and higher lipid yields were observed. To understand this phenomenon, RNA-seq analysis was implemented to study the gene expression profiles during growth on mixtures mainly to elucidate regulation of glycerol metabolism. Insights into lipid biosynthesis on mixed substrates are provided at a systems level. Among others, transcriptional profiles showed that glycerol might be produced intracellularly and glycerol kinase (GUT1) and glycerol 3-phosphate dehydrogenase (GUT2) enzymes were not downregulated in the presence of glucose. Transcriptional analysis also showed that the regulation of glycerol uptake in the presence of glucose at transcriptional level is different from that observed in Saccharomyces cerevisiae.

Metabolic network analysis and experimental study of lipid production in Rhodosporidium toruloides grown on single and mixed substrates.

BACKGROUND: Microbial lipids (triacylglycerols, TAG) have received large attention for a sustainable production of oleochemicals and biofuels. Rhodosporidium toruloides can accumulate lipids up to 70% of its cell mass under certain conditions. However, our understanding of lipid production in this yeast is still much limited, especially for growth with mixed substrates at the level of metabolic network. In this work, the potentials of several important carbon sources for TAG production in R.toruloides are first comparatively studied in silico by means of elementary mode analysis followed by experimental validation. RESULTS: A simplified metabolic network of R.toruloides was reconstructed based on a combination of genome and proteome annotations. Optimal metabolic space was studied using elementary mode analysis for growth on glycerol, glucose, xylose and arabinose or in mixtures. The in silico model predictions of growth and lipid production are in agreement with experimental results. Both the in silico and experimental studies revealed that glycerol is an attractive substrate for lipid synthesis in R. toruloides either alone or in blend with sugars. A lipid yield as high as 0.53 (C-mol TAG/C-mol) has been experimentally obtained for growth on glycerol, compared to a theoretical maximum of 0.63 (C-mol TAG/C-mol). The lipid yield on glucose is much lower (0.29 (experimental) vs. 0.58 (predicted) C-mol TAG/C-mol). The blend of glucose with glycerol decreased the lipid yield on substrate but can significantly increase the overall volumetric productivity. Experimental studies revealed catabolite repression of glycerol by the presence of glucose for the first time. Significant influence of oxygen concentration on the yield and composition of lipids were observed which have not been quantitatively studied before. CONCLUSIONS: This study provides for the first time a simplified metabolic model of R.toruloides and its detailed in silico analysis for growth on different carbon sources for their potential of TAG synthesis. Experimental studies revealed the phenomenon of catabolite repression of glycerol by glucose and the importance of oxygen supply on the yield and composition of lipids. More systematic studies are needed to understand the mechanisms which should help to further optimize the lipid production in this strain of industrial interest.

A de novo NADPH generation pathway for improving lysine production of Corynebacterium glutamicum by rational design of the coenzyme specificity of glyceraldehyde 3-phosphate dehydrogenase.

Engineering the cofactor availability is a common strategy of metabolic engineering to improve the production of many industrially important compounds. In this work, a de novo NADPH generation pathway is proposed by altering the coenzyme specificity of a native NAD-dependent glyceraldehyde 3-phosphate dehydrogenase (GAPDH) to NADP, which consequently has the potential to produce additional NADPH in the glycolytic pathway. Specifically, the coenzyme specificity of GAPDH of Corynebacterium glutamicum is systematically manipulated by rational protein design and the effect of the manipulation for cellular metabolism and lysine production is evaluated. By a combinatorial modification of four key residues within the coenzyme binding sites, different GAPDH mutants with varied coenzyme specificity were constructed. While increasing the catalytic efficiency of GAPDH towards NADP enhanced lysine production in all of the tested mutants, the most significant improvement of lysine production (~60%) was achieved with the mutant showing similar preference towards both NAD and NADP. Metabolic flux analysis with (13)C isotope studies confirmed that there was no significant change of flux towards the pentose phosphate pathway and the increased lysine yield was mainly attributed to the NADPH generated by the mutated GAPDH. The present study highlights the importance of protein engineering as a key strategy in de novo pathway design and overproduction of desired products.

Deregulation of feedback inhibition of phosphoenolpyruvate carboxylase for improved lysine production in Corynebacterium glutamicum.

Allosteric regulation of phosphoenolpyruvate carboxylase (PEPC) controls the metabolic flux distribution of anaplerotic pathways. In this study, the feedback inhibition of Corynebacterium glutamicum PEPC was rationally deregulated, and its effect on metabolic flux redistribution was evaluated. Based on rational protein design, six PEPC mutants were designed, and all of them showed significantly reduced sensitivity toward aspartate and malate inhibition. Introducing one of the point mutations (N917G) into the ppc gene, encoding PEPC of the lysine-producing strain C. glutamicum LC298, resulted in ∼37% improved lysine production. In vitro enzyme assays and (13)C-based metabolic flux analysis showed ca. 20 and 30% increases in the PEPC activity and corresponding flux, respectively, in the mutant strain. Higher demand for NADPH in the mutant strain increased the flux toward pentose phosphate pathway, which increased the supply of NADPH for enhanced lysine production. The present study highlights the importance of allosteric regulation on the flux control of central metabolism. The strategy described here can also be implemented to improve other oxaloacetate-derived products.

Novel (2R,3R)-2,3-butanediol dehydrogenase from potential industrial strain Paenibacillus polymyxa ATCC 12321.

A (2R,3R)-2,3-butanediol dehydrogenase (BDH99::67) from Paenibacillus polymyxa ATCC 12321 was functionally characterized. The genetic characteristics of BDH99::67 are completely different from those of meso- and (2S,3S)-2,3-butanediol dehydrogenases. The results showed that BDH99::67 belongs to the medium-chain dehydrogenase/reductase superfamily and not to the short-chain dehydrogenase/reductase superfamily, to which meso- and (2S,3S)-2,3-butanediol dehydrogenases belong.