Biotechnology for Biofuels最新文献

Background

The phenolic compound tyrosol is widely used in the pharmaceutical industry, owing to its beneficial effects on human health and its use as a precursor for key pharmaceuticals, including β₁-receptor blockers. Tyrosol can be found in olive oil, but despite its natural biosynthesis in plants, low extraction efficiencies render microbial production a more viable alternative.

Results

Here, we engineered the l-tyrosine overproducing Corynebacterium glutamicum strain AROM3 for the de novo production of tyrosol. Two routes were established and compared: one via 4-OH-phenylpyruvate as intermediate and the other via tyramine. We initially expected the first route to require heterologous expression of a prephenate dehydrogenase gene, given that C. glutamicum lacks this enzymatic function. However, heterologous expression of ARO10 from Saccharomyces cerevisiae (ARO10_Sc), which encodes a phenylpyruvate decarboxylase, was sufficient to establish tyrosol production in strain AROM3. We identified that 4-OH-phenylpyruvate is synthesized from l-tyrosine by native aminotransferases, which is subsequently decarboxylated by Aro10_Sc, and reduced to tyrosol by native alcohol dehydrogenases, leading to a titer of 9.4 ± 1.1 mM (1.30 ± 0.15 g/L). We identified the furfural dehydrogenase FudC as major enzyme involved in this pathway, as its gene deletion reduced tyrosol production by 75%. Given the instability of 4-OH-phenylpyruvate, the synthesis of tyrosol via the stable intermediate tyramine was pursued via the second route. Decarboxylation of l-tyrosine followed by oxidative deamination was accomplished by overexpression of the l-tyrosine decarboxylase gene tdc from Levilactobacillus brevis (tdc_Lb) and the tyramine oxidase gene tyo from Kocuria rhizophila (tyo_Kr). Using this route, tyrosol production was increased by 44% compared to the route via 4-OH-phenylpyruvate. With a division of labor approach by co-cultivating l-tyrosine producing strains that either express tdc_Lb or tyo_Kr, the highest titer of 14.1 ± 0.3 mM (1.95 ± 0.04 g/L) was achieved.

Conclusions

This study demonstrates the potential of endotoxin-free C. glutamicum as production host for the l-tyrosine-derived product tyrosol. Due to its l-arogenate pathway for l-tyrosine synthesis, the unstable 4-OH-phenylpyruvate could be excluded as intermediate in the Tdc–Tyo pathway, outcompeting the most often utilized production route via phenylpyruvate decarboxylases.

Background

The global energy crisis, driven by economic growth and the increasing demand for energy, highlights the urgency of searching for alternative energy sources to mitigate environmental pollution and climate change. β-Glucosidases act in the final step of the enzymatic hydrolysis of cellulose, cleaving the β-1,4-glycosidic bonds in cellobiose to produce second-generation ethanol. However, these enzymes are easily inhibited by glucose, their final product, which limits the production of this biofuel. Genetic engineering combined with bioinformatics tools can improve key enzymatic characteristics, such as catalytic activity and glucose tolerance, in a more precise, faster, and cost-effective manner compared to traditional methods. In this work, a variant of a β-glucosidase from the GH1 family, isolated from the microbial community of Amazonian soil (Brazil), with enhanced catalytic activity and improved for glucose retroinhibition, was developed.

Results

Bioinformatics analyses suggested the substitution of tryptophan at position 404 with leucine. The produced variant (W404L) was expressed in Escherichia coli and showed activity 3.2 times higher in the presence of glucose than the non-mutated control. Moreover, the partially purified mutated variant of β-glucosidase exhibited a 26-fold increase in catalytic activity compared to the original form of the enzyme. The results confirmed that the mutation proposed by computational analyses had a significant impact on enzyme catalytic activity and glucose retroinhibition.

Conclusions

This new variant may become a promising alternative to reduce the costs of enzyme cocktails used in the hydrolysis of lignocellulosic biomass used as a raw material in the production of second-generation ethanol.

Graphical Abstract

Background

Pharmaceutical safety is an increasing global priority, particularly as the demand for therapeutic compounds rises alongside population growth. Phytocannabinoids, a class of bioactive polyketide molecules derived from plants, have garnered significant attention due to their interaction with the human endocannabinoid system, offering potential benefits for managing a range of symptoms and conditions. Traditional extraction from cannabis plants poses regulatory, environmental, and yield-related challenges. Consequently, microbial biosynthesis has emerged as a promising biotechnological alternative to produce cannabinoids in a controlled, scalable, and sustainable manner. Developing diatom-based biofactories represent a crucial step in advancing this biotechnology, enabling the efficient production of high-valued compounds such as cannabinoids.

Results

We engineered the diatom Phaeodactylum tricornutum, a unicellular photosynthetic model organism prized for its naturally high lipid content, to produce olivetolic acid (OA), a key metabolic precursor to most cannabinoids. The genes encoding tetraketide synthase and olivetolic acid cyclase from cannabis were cloned onto episomal vectors and introduced using bacterial conjugation in two separate P. tricornutum transconjugant lines to evaluate enzyme activity and OA production in vivo. Both genes were successfully expressed, and the corresponding enzymes accumulated within the transconjugant lines. However, despite testing the cell extracts individually and in combination, OA accumulation was not detected suggesting potential conversion or utilization of OA by endogenous metabolic pathways within the diatoms. To investigate this further, we analyzed the impact of CsTKS expression on the diatom’s metabolome, revealing significant alterations that may indicate metabolic flux redirection or novel pathway interactions.

Conclusions

Our study demonstrates the successful expression of cannabinoid biosynthetic genes in P. tricornutum but highlights challenges in OA accumulation, likely due to endogenous metabolic interactions. These findings underscore the complexity of metabolic engineering in diatoms and suggest the need for further pathway optimization and metabolic flux analysis to achieve efficient cannabinoid biosynthesis. This research contributes to advancing sustainable biotechnological approaches for cannabinoid production.

Graphical abstract

Cyanobacteria bear great biotechnological potential as photosynthetic cell factories. In particular, hydrogenases are promising with respect to light-driven H₂ production as well as H₂-driven redox biocatalysis. Their utilization relies on effective strain design as well as a balanced synthesis and maturation of heterologous enzymes. In a previous study, the soluble O₂-tolerant hydrogenase complex from Cupriavidus necator (CnSH) could be introduced into the model cyanobacterium Synechocystis sp. PCC 6803. Due to its O₂-tolerance, it was indeed active under photoautotrophic growth conditions. However, the specific activity was rather low indicating that further engineering is required, for which we followed a two-step approach. First, we optimized the CnSH multigene expression in Synechocystis by applying different regulatory elements. Although corresponding protein levels and specific CnSH activity increased, the apparent rise in enzyme levels did not fully translate into activity increase. Second, the entire set of hyp genes, encoding CnSH maturases, was co-expressed in Synechocystis to investigate, if CnSH maturation was limiting. Indeed, the native CnSH maturation apparatus promoted functional CnSH synthesis, enabling a threefold higher H₂ oxidation activity compared to the parental strain. Our results suggest that a fine balance between heterologous hydrogenase and maturase expression is required to ensure high specific activity over an extended time period.

To address the challenge of microbial tolerance in industrial biomanufacturing, we developed an adaptive evolution strategy for Escherichia coli W3110 to enhance its salicylic acid (SA) tolerance. Utilizing a CmeR-P_cmeO biosensor-enabled high-throughput screening system, we isolated an SA-tolerant variant (W3110K-4) that exhibited a 2.3-fold increase in tolerance (from 0.9 to 2.1 g/L) and a 2.1-fold improvement in SA production (from 283 to 588.1 mg/L). Subsequently, the designed sensors were combined with multi-pathway sgRNA arrays to dynamically modulate the other three branched-chain acid derivatives, achieving a balance between biomass growth and rapid SA production in the adaptively evolved strain, resulting in a maximum SA yield of 1477.8 mg/L, which represents a 30% improvement over the non-evolved control strain W3110K-W2 (1138.2 mg/L) using the same metabolic strategy. Whole-genome sequencing revealed that adaptive mutations in genes such as ducA* and anti-drug resistance C2 mutation genes (ymdA*, ymdB*, clsC*, csgB*, csgA*, and csgC*) play a key role in enhancing SA tolerance and productivity. Notably, the evolved strain W3110K-4 exhibits significant resistance to bacteriophages, making it a promising candidate for large-scale SA fermentation. This work develops and expands the CmeR-P_cmeO system, proposes new insights into improved strains through biosensor screening, guided multi-pathway metabolism, and adaptive evolution, and provides a paradigm for engineers to obtain engineered strains.

Background

Dunaliella microalgae, such as Dunaliella salina riching in β-carotene and Dunaliella bardawil rich in lutein and α-carotene, have been used in aquaculture, supplements, cosmetics, and feed industries. The genus Dunaliella is diverse; therefore, characterization of novel strains and isolation of new varieties through mutagenesis technology will promote natural carotenoid bioproduction.

Results

Salt stress test demonstrated that the newly isolated microalgae strain ZP-1 was a halotolerant strain. Morphology observation and molecular phylogeny analysis indicated that the unicellular green microalga ZP-1 was a member of the genus Dunaliella. Biomass of ZP-1 in RAM medium was up to 2.45 g/L, showing the advantage over other common Dunaliella microalgae in terms of yield. Furthermore, Ethyl methanesulfonate (EMS) mutant library was generated from this high-biomass strain, aiming to improve natural carotenoid productivity. A mutant strain was selected through morphology observation combining with carotenoid quantification by HPLC, which was nominated as turn yellow dunaliella 4 (tyd4). The mutant tyd4 displayed an increased lutein productivity by 28.55% and an increased zeaxanthin productivity by 22.19%. Biomass of tyd4 was promoted by 17.40% through continuous culture under red light. Application of exogenous 1.0 μM melatonin on the mutant tyd4 led to increased cell density and improved biomass.

Conclusions

Results in this study support that EMS mutagenesis is an effective breeding approach for further improvement of Dunaliella sp. ZP-1, which is a high-biomass microalgae exhibiting potential to overcome the bottleneck of low biomass of current commercial Dunaliella strains. The mutant tyd4 had higher contents of both lutein and zeaxanthin, whose yield could be further elevated by red light and melatonin. This study provided new microalgae sources for scientific research and technical reference for the bioproduction of natural carotenoids.

l-threonine is an integral nutrient for mammals, often used in animal feeds to enhance growth and reduce breeding costs. Developing l-threonine engineered strains that meet industrial production specifications has significant economic value. Here, we developed a biosensor that monitors l-threonine concentration to assist in high-throughput screening to capture high-yielding l-threonine mutants. Among them, the P_cysK promoter and CysB protein were used to construct a primary l-threonine biosensor, and then the CysB^T102A mutant was obtained through directed evolution resulting in a 5.6-fold increase in the fluorescence responsiveness of biosensor over the 0–4 g/L l-threonine concentration range. In addition, the metabolic network of mutant was further optimized through multi-omics analysis and in silico simulation. Ultimately, the THRM13 strain produced 163.2 g/L l-threonine, with a yield of 0.603 g/g glucose in a 5 L bioreactor. The biosensor constructed here could be employed for iterative upgrading of subsequent strains, and these engineering strategies described provide guidance for other chemical overproducers.

Graphical Abstract

Background

Lignin–carbohydrate complexes in lignocellulosic biomass act as a barrier to its biodegradation and biotechnological exploitation. Enzymatic dissociation between lignin and hemicellulose is a key process that allows the efficient bioconversion of both polymers. Glucuronoyl esterases of the Carbohydrate Esterase 15 family target the ester linkages between the glucuronic acid of xylan and lignin moieties, assisting enzymatic biodegradation of lignocellulose.

Results

In this study, two CE15 glucuronoyl esterases from the white-rot fungi Artolenzites elegans and Trametes ljubarskyi were heterologously expressed in Pichia pastoris and biochemically characterized on the model substrate D-glucuronic acid ester with cinnamyl alcohol and a variety of pretreated lignocellulosic biomasses. The pretreatment method was shown to be a determining factor in revealing both the activity of the esterases on lignocellulose and their synergistic relationships with other hemicellulases. AeGE15 and TlGE15 demonstrated activity on pretreated biomass with high hemicellulose and lignin content, increasing saccharification by 57 ± 1 μM and 61 ± 3 μM of xylose equivalents, respectively. Furthermore, the synergy between these CE15 esterases and three xylanases from distinct glycoside hydrolase families (GH10, GH11 and GH30) was investigated on pretreated lignocellulosic samples, highlighting beneficial enzymatic interplays. Pretreated birchwood degradation by AnXyn11 was increased from 6% to approximately 10% by the esterases, based on xylose equivalents of unsubstituted xylooligomers. The GEs also promoted the glucuronoxylanase specificity of TtXyn30A, leading up to three-times higher release in aldouronic acids. Finally, a synergistic effect between AeGE15 and TmXyn10 was observed on pretreated corn bran, increasing xylose and xylotriose release by 27 ± 8% and 55 ± 15%, respectively.

Conclusions

Both CE15 esterases promoted biomass saccharification by the xylanases, while there was a prominent effect on the GH30 glucuronoxylanase regarding the release of aldouronic acids. Overall, this study shed some light on the role of CE15 glucuronoyl esterases in the enzymatic biodegradation of plant biomass, particularly its (arabino)glucuronoxylan component, during cooperative activity with xylanases.

Background

Biofuels produced from algae have enormous advantages in replacing fossil fuels, and Microcystis aeruginosa has a great potential for biofuel production, due to growing fast to form large amounts of biomass. Light is essential for algal growth, and the optimum light quality can promote the biomass and lipid accumulation for increasing feedstock for biofuel production.

Results

We investigated the biomass accumulation, photosynthetic ability, carbohydrate, and lipid yield as well as related gene expression in M. aeruginosa under red, blue, purple, and white light to promote biofuel production using this alga under the optimal light quality. Compared with white light, purple light promoted the cell growth during the 5 days, while blue light showed inhibitory effect. Red light had no effect on the cell growth, but improved the biomass content to the highest level. Red light improved the photosynthetic ability by raising chlorophyll level, and up-regulating expression of the genes in chlorophyll biosynthesis, photosynthetic electron transfer, and CO₂ fixation. Among these light qualities, red light showed the maximum effect on soluble, insoluble, and total carbohydrate accumulation by up-regulating the genes in polysaccharide and starch formation, and down-regulating the genes in glycolysis and tricarboxylic acid cycle. Red light also exhibited the maximum effect on lipid accumulation, which might be caused by up-regulating five genes in fatty acid biosynthesis.

Conclusion

Red light can promote M. aeruginosa accumulating carbohydrates and lipids by regulating related gene expression, which should be the optimal light quality for improving feedstock yield for biofuel production.

Background

Oil from oleaginous yeasts has the potential to replace non-sustainable vegetable oil as raw material to produce food, feed, biofuels, or biochemicals. Co-produced compounds like carotenoids may be helpful to obtain economically viable bioprocesses. Identifying appropriate extraction methods is a bottleneck both for establishing oleaginous yeasts as cell factories for both oil and carotenoids production and for analysis of intracellular compounds like lipids and carotenoids. We conducted extractions using supercritical carbon dioxide (SC-CO₂) and conventional solvent methods to extract and analyze lipids and carotenoids from R. toruloides CBS 14 cells grown on wheat straw hydrolysate. The lipid extracts were analyzed using gas chromatography (GC), and the carotenoids were identified and quantified using ultra-high-performance liquid chromatography (UHPLC).

Results

Four main carotenoids in the extracts from both extraction methods were identified including β-carotene, γ-carotene, torularhodin, and torulene. Interestingly, torularhodin was the major carotenoid extracted using SC-CO₂ extraction, followed by torulene. This was different from the conventional acetone extraction method, where β-carotene was the main carotenoid. After the conventional extraction, torularhodin and torulene underwent degradation due to the saponification step, which was necessary to remove lipids before UHPLC analysis. The total carotenoid concentration obtained from SC-CO₂ extraction was 332.09 ± 27.32 μg/g dry weight compared to 19.9 ± 2.74 μg/g dry weight in acetone extraction. A small amount of carotenoids was observed to be lost into the lipid extract, but this loss was not as substantial as that seen with acetone extraction. Additionally, the total lipid content in samples extracted using SC-CO₂ was significantly lower than that obtained using the conventional Folch method. GC analysis revealed that oleic acid was the major fatty acid in both lipid extracts, followed by palmitic acid and linoleic acid. Notably, the proportion of unsaturated fatty acids was higher in the extracts from the SC-CO₂ method compared to the conventional method.

Conclusion

These findings indicate that the SC-CO₂ extraction method outperformed conventional methods by preserving the integrity of unsaturated lipids and retaining an abundance of carotenoids, resulting in high-quality extracts.