Biotechnology for Biofuels and Bioproducts最新文献

Background: Artificial microbial consortia composed of heterotrophic and photoautotrophic organisms represent a unique strategy for converting light energy and carbon dioxide into high-value bioproducts. Currently, the types of desired bioproducts are still limited, and microbial fitness benefit rendered by paired partner generally needs to be intensified. Exploring novel artificial microbial consortia at a laboratory scale is an essential step towards addressing this unmet need. This study aimed to conduct and analyze an artificial consortium composed of cyanobacterium Synechococcus elongatus FL130 with the filamentous fungus Aspergillus nidulans TWY1.1 for producing fungi-derived secondary metabolite of polyketide neosartoricin B.

Results: Polyketide-producing A. nidulans TWY1.1 substantially ameliorated the growth and the survival of sucrose-secreting cyanobacterium S. elongatus FL130 in salt-stressed environments. Besides sucrose, comparable amounts of other carbohydrates were released from axenically cultured FL130 cells, which could be efficiently consumed by TWY1.1. Relative to axenically cultured FL130, less glycogen was accumulated in FL130 cells co-cultured with TWY1.1, and the glycogen phosphorylase gene catalyzing the first step for glycogen degradation had two-fold expression. Different from axenically cultured filamentous fungi, abundant vacuoles were observed in fungal hyphae of TWY1.1 co-cultured with cyanobacterium FL130. Meanwhile, FL130 cells displayed a characteristic pattern of interacting with its heterotrophic partner, densely dispersing along certain hyphae of TWY1.1. Finally, polyketide neosartoricin B was produced from TWY1.1 in FL130-TWY1.1 co-cultures, which was tightly adjusted by nitrogen level.

Conclusion: Overall, the results thoroughly proved the concept of pairing cyanobacteria with filamentous fungi to build artificial consortia for producing fungi-derived biomolecules.

Background: Heme has attracted much attention because of its wide applications in medicine and food. The products of genes hemBCDEFY convert 5-aminolevulinic acid to protoporphyrin IX (PPIX; the immediate precursor of heme); protoporphyrin ferrochelatase (FECH) inserts Fe²⁺ into PPIX to generate heme. Biosynthesis of heme is limited by the need for optimized expression levels of multiple genes, complex regulatory mechanisms, and low enzymatic activity; these problems need to be overcome in metabolic engineering to improve heme synthesis.

Results: We report a heme biosensor-guided screening strategy using the heme-responsive protein HrtR to regulate tcR expression in Escherichia coli, providing a quantifiable link between the intracellular heme concentration and cell survival in selective conditions (i.e., the presence of tetracycline). This system was used for rapid enrichment screening of heme-producing strains from a library with random ribosome binding site (RBS) variants and from a FECH mutant library. Through up to four rounds of iterative evolution, strains with optimal RBS intensities for the combination of hemBCDEFY were screened; we obtained a PPIX titer of 160.8 mg/L, the highest yield yet reported in shaken-flask fermentation. A high-activity FECH variant was obtained from the saturation mutagenesis library. Fed-batch fermentation of strain SH20C, harboring the optimized hemBCDEFY and the FECH mutant, produced 127.6 mg/L of heme.

Conclusion: We sequentially improved the multigene biosynthesis pathway of PPIX and performed in vivo directed evolution of FECH, based on a heme biosensor, which demonstrated the effectiveness of the heme biosensor-based pathway optimization strategy and broadens our understanding of the mechanism of heme synthesis.

Background: 5-Aminolevulinic acid (5-ALA) is a promising biostimulant, feed nutrient, and photodynamic drug with wide applications in modern agriculture and therapy. Although microbial production of 5-ALA has been improved realized by using metabolic engineering strategies during the past few years, there is still a gap between the present production level and the requirement of industrialization.

Results: In this study, pathway, protein, and cellular engineering strategies were systematically employed to construct an industrially competitive 5-ALA producing Escherichia coli. Pathways involved in precursor supply and product degradation were regulated by gene overexpression and synthetic sRNA-based repression to channel metabolic flux to 5-ALA biosynthesis. 5-ALA synthase was rationally engineered to release the inhibition of heme and improve the catalytic activity. 5-ALA transport and antioxidant defense systems were targeted to enhance cellular tolerance to intra- and extra-cellular 5-ALA. The final engineered strain produced 30.7 g/L of 5-ALA in bioreactors with a productivity of 1.02 g/L/h and a yield of 0.532 mol/mol glucose, represent a new record of 5-ALA bioproduction.

Conclusions: An industrially competitive 5-ALA producing E. coli strain was constructed with the metabolic engineering strategies at multiple layers (protein, pathway, and cellular engineering), and the strategies here can be useful for developing industrial-strength strains for biomanufacturing.

Background: Escherichia coli cells are the most frequently used hosts in recombinant protein production processes and mainly require molecules such as IPTG or pure lactose as inducers of heterologous expression. A possible way to reduce the production costs is to replace traditional inducers with waste materials such as cheese whey permeate (CWP). CWP is a secondary by-product generated from the production of the valuable whey proteins, which are obtained from ultrafiltration of cheese whey, a main by-product of the dairy industry, which is rich in lactose.

Results: The effects of CWP collected from an Italian plant were compared with those of traditional inducers on the production of two model proteins (i.e., green fluorescent protein and the toxic Q55 variant of ataxin-3), in E. coli BL21 (DE3) cells. It was found that the high lactose content of CWP (165 g/L) and the antioxidant properties of its micronutrients (vitamins, cofactors and osmolytes) sustain production yields similar to those obtained with traditional inducers, accompanied by the improvement of cell fitness.

Conclusions: CWP has proven to be an effective and low-cost alternative inducer to produce recombinant proteins. Its use thus combines the advantage of exploiting a waste product with that of reducing the production costs of recombinant proteins.

Background: Perennial C₄ grasses from the genus Miscanthus are widely regarded as leading and promising dedicated bioenergy crops due to their high biomass accumulation on marginal land with low environmental impacts and maintenance requirements over its productive life. There is an urgent socio-political and environmental need to ramp up the production of alternative, affordable and green bioenergy sources and to re-direct the net zero carbon emissions trajectory. Hence, up-scaling of Miscanthus cultivation as a source of biomass for renewable energy could play an important role to strategically address sustainable development goals for a growing bio-based economy. Certain Miscanthus sinensis genotypes are particularly interesting for their biomass productivity across a wide range of locations. As the aromatic biomass component lignin exhibits a higher energy density than cell wall polysaccharides and is generally used as an indicator for heating or calorific value, genetic engineering could be a feasible strategy to develop M. sinensis biomass with increased lignin content and thus improving the energetic value of the biomass.

Results: For this purpose, transgenic M. sinensis were generated by Agrobacterium-mediated transformation for expression of ZmMYB167, a MYB transcription factor known for regulating lignin biosynthesis in C₃ and C₄ grasses. Four independent transgenic ZmMYB167 Miscanthus lines were obtained. Agronomic traits such as plant height, tillering and above-ground dry weight biomass of the transgenic plants were not different to that of wild-type control plants. Total lignin content of the transgenic plants was ~ 15-24% higher compared with control plants. However, the structural carbohydrates, glucan and xylan, were decreased by ~ 2-7% and ~ 8-10%, respectively, in the transgenic plants. Moreover, expression of ZmMYB167 in transgenic plants did not alter lignin composition, phenolic compounds or enzymatic saccharification efficiency yields but importantly improved total energy levels in Miscanthus biomass, equivalent to 10% higher energy yield per hectare.

Conclusions: This study highlights ZmMYB167 as a suitable target for genetic lignin bioengineering interventions aimed at advancing and developing lignocellulosic biomass supply chains for sustainable production of renewable bioenergy.

Background: Microalgae represent a suitable and eco-sustainable resource for human needs thanks to their fast growth ability, together with the great diversity in species and intracellular secondary bioactive metabolites. These high-added-value compounds are of great interest for human health or animal feed. The intracellular content of these valuable compound families is tightly associated with the microalgal biological state and responds to environmental cues, e.g., light. Our study develops a Biotechnological response curve strategy exploring the bioactive metabolites synthesis in the marine cyanobacterium Spirulina subsalsa over a light energy gradient. The Relative Light energy index generated in our study integrates the red, green and blue photon flux density with their relative photon energy. The Biotechnological response curve combined biochemical analysis of the macromolecular composition (total protein, lipid, and carbohydrate content), total sterols, polyphenols and flavonoids, carotenoids, phenolic compounds, vitamins (A, B₁, B₂, B₆, B₉, B₁₂, C, D₂, D₃, E, H, and K₁), phycobiliproteins, together with the antioxidant activity of the biomass as well as the growth ability and photosynthesis.

Results: Results demonstrated that light energy significantly modulate the biochemical status of the microalga Spirulina subsalsa revealing the relevance of the light energy index to explain the light-induced biological variability. The sharp decrease of the photosynthetic rate at high light energy was accompanied with an increase of the antioxidant network response, such as carotenoids, total polyphenols, and the antioxidant capacity. Conversely, low light energy favorized the intracellular content of lipids and vitamins (B₂, B₆, B₉, D₃, K₁, A, C, H, and B₁₂) compared to high light energy.

Conclusions: Results of the Biotechnological response curves were discussed in their functional and physiological relevance as well as for the essence of their potential biotechnological applications. This study emphasized the light energy as a relevant tool to explain the biological responses of microalgae towards light climate variability, and, therefore, to design metabolic manipulation of microalgae.

Background: Elevated CO₂ partial pressure (pCO₂) has been proposed as a potential steering parameter for selective carboxylate production in mixed culture fermentation. It is anticipated that intermediate product spectrum and production rates, as well as changes in the microbial community, are (in)directly influenced by elevated pCO₂. However, it remains unclear how pCO₂ interacts with other operational conditions, namely substrate specificity, substrate-to-biomass (S/X) ratio and the presence of an additional electron donor, and what effect pCO₂ has on the exact composition of fermentation products. Here, we investigated possible steering effects of elevated pCO₂ combined with (1) mixed substrate (glycerol/glucose) provision; (2) subsequent increments in substrate concentration to increase the S/X ratio; and (3) formate as an additional electron donor.

Results: Metabolite predominance, e.g., propionate vs. butyrate/acetate, and cell density, depended on interaction effects between pCO₂-S/X ratio and pCO₂-formate. Individual substrate consumption rates were negatively impacted by the interaction effect between pCO₂-S/X ratio and were not re-established after lowering the S/X ratio and adding formate. The product spectrum was influenced by the microbial community composition, which in turn, was modified by substrate type and the interaction effect between pCO₂-formate. High propionate and butyrate levels strongly correlated with Negativicutes and Clostridia predominance, respectively. After subsequent pressurized fermentation phases, the interaction effect between pCO₂-formate enabled a shift from propionate towards succinate production when mixed substrate was provided.

Conclusions: Overall, interaction effects between elevated pCO₂, substrate specificity, high S/X ratio and availability of reducing equivalents from formate, rather than an isolated pCO₂ effect, modified the proportionality of propionate, butyrate and acetate in pressurized mixed substrate fermentations at the expense of reduced consumption rates and increased lag-phases. The interaction effect between elevated pCO₂ and formate was beneficial for succinate production and biomass growth with a glycerol/glucose mixture as the substrate. The positive effect may be attributed to the availability of extra reducing equivalents, likely enhanced carbon fixating activity and hindered propionate conversion due to increased concentration of undissociated carboxylic acids.

Background: Production of monocarboxylates using microbial communities is highly dependent on local and degradable biomass feedstocks. Syngas or different mixtures of H₂, CO, and CO₂ can be sourced from biomass gasification, excess renewable electricity, industrial off-gases, and carbon capture plants and co-fed to a fermenter to alleviate dependence on local biomass. To understand the effects of adding these gases during anaerobic fermentation of plant biomass, a series of batch experiments was carried out with different syngas compositions and corn silage (pH 6.0, 32 °C).

Results: Co-fermentation of syngas with corn silage increased the overall carboxylate yield per gram of volatile solids (VS) by up to 29% (0.47 ± 0.07 g g_VS^-1; in comparison to 0.37 ± 0.02 g g_VS^-1 with a N₂/CO₂ headspace), despite slowing down biomass degradation. Ethylene and CO exerted a synergistic effect in preventing methanogenesis, leading to net carbon fixation. Less than 12% of the electrons were misrouted to CH₄ when either 15 kPa CO or 5 kPa CO + 1.5 kPa ethylene was used. CO increased the selectivity to acetate and propionate, which accounted for 85% (electron equivalents) of all products at 49 kPa CO, by favoring lactic acid bacteria and actinobacteria over n-butyrate and n-caproate producers. Inhibition of n-butyrate and n-caproate production by CO happened even when an inoculum preacclimatized to syngas and lactate was used. Intriguingly, the effect of CO on n-butyrate and n-caproate production was reversed when formate was present in the broth.

Conclusions: The concept of co-fermenting syngas and plant biomass shows promise in three aspects: by making anaerobic fermentation a carbon-fixing process, by increasing the yields of short-chain carboxylates (propionate and acetate), and by minimizing electron losses to CH₄. Moreover, a model was proposed for how formate can alleviate CO inhibition in certain acidogenic bacteria. Testing the fermentation of syngas and plant biomass in a continuous process could potentially improve selectivity to n-butyrate and n-caproate by enriching chain-elongating bacteria adapted to CO and complex biomass.

Background: Co-cultures and cell immobilization have been used for retaining biomass in a bioreactor, with the aim to improve the volumetric hydrogen productivity (Q_H2). Caldicellulosiruptor kronotskyensis is a strong cellulolytic species that possesses tāpirin proteins for attaching on lignocellulosic materials. C. owensensis has its reputation as a biofilm former. It was investigated whether continuous co-cultures of these two species with different types of carriers can improve the Q_H2.

Results: Q_H2 up to 30 ± 0.2 mmol L^-1 h^-1 was obtained during pure culture of C. kronotskyensis with combined acrylic fibres and chitosan. In addition, the yield of hydrogen was 2.95 ± 0.1 mol H₂ mol^-1 sugars at a dilution rate (D) of 0.3 h^-1. However, the second-best Q_H2 26.4 ± 1.9 mmol L^-1 h^-1 and 25.4 ± 0.6 mmol L^-1 h^-1 were obtained with a co-culture of C. kronotskyensis and C. owensensis with acrylic fibres only and a pure culture of C. kronotskyensis with acrylic fibres, respectively. Interestingly, the population dynamics revealed that C. kronotskyensis was the dominant species in the biofilm fraction, whereas C. owensensis was the dominant species in the planktonic phase. The highest amount of c-di-GMP (260 ± 27.3 µM at a D of 0.2 h^-1) were found with the co-culture of C. kronotskyensis and C. owensensis without a carrier. This could be due to Caldicellulosiruptor producing c-di-GMP as a second messenger for regulation of the biofilms under the high dilution rate (D) to prevent washout.

Conclusions: The cell immobilization strategy using a combination of carriers exhibited a promising approach to enhance the Q_H2. The Q_H2 obtained during the continuous culture of C. kronotskyensis with combined acrylic fibres and chitosan gave the highest Q_H2 among the pure culture and mixed cultures of Caldicellulosiruptor in the current study. Moreover, it was the highest Q_H2 among all cultures of Caldicellulosiruptor species studied so far.

Background: Developing new bioprocesses to produce chemicals and fuels with reduced production costs will greatly facilitate the replacement of fossil-based raw materials. In most fermentation bioprocesses, the feedstock usually represents the highest cost, which becomes the target for cost reduction. Additionally, the biorefinery concept advocates revenue growth from the production of several compounds using the same feedstock. Taken together, the production of bio commodities from low-cost gas streams containing CO, CO₂, and H₂, obtained from the gasification of any carbon-containing waste streams or off-gases from heavy industry (steel mills, processing plants, or refineries), embodies an opportunity for affordable and renewable chemical production. To achieve this, by studying non-model autotrophic acetogens, current limitations concerning low growth rates, toxicity by gas streams, and low productivity may be overcome. The Acetobacterium wieringae strain JM is a novel autotrophic acetogen that is capable of producing acetate and ethanol. It exhibits faster growth rates on various gaseous compounds, including carbon monoxide, compared to other Acetobacterium species, making it potentially useful for industrial applications. The species A. wieringae has not been genetically modified, therefore developing a genetic engineering method is important for expanding its product portfolio from gas fermentation and overall improving the characteristics of this acetogen for industrial demands.

Results: This work reports the development and optimization of an electrotransformation protocol for A. wieringae strain JM, which can also be used in A. wieringae DSM 1911, and A. woodii DSM 1030. We also show the functionality of the thiamphenicol resistance marker, catP, and the functionality of the origins of replication pBP1, pCB102, pCD6, and pIM13 in all tested Acetobacterium strains, with transformation efficiencies of up to 2.0 × 10³ CFU/μg_DNA. Key factors affecting electrotransformation efficiency include OD₆₀₀ of cell harvesting, pH of resuspension buffer, the field strength of the electric pulse, and plasmid amount. Using this method, the acetone production operon from Clostridium acetobutylicum was efficiently introduced in all tested Acetobacterium spp., leading to non-native biochemical acetone production via plasmid-based expression.

Conclusions: A. wieringae can be electrotransformed at high efficiency using different plasmids with different replication origins. The electrotransformation procedure and tools reported here unlock the genetic and metabolic manipulation of the biotechnologically relevant A. wieringae strains. For the first time, non-native acetone production is shown in A. wieringae.