Biotechnology for Biofuels and Bioproducts最新文献

Background: Microbial electricity production has received considerable attention from researchers due to its environmental friendliness and low price. The increase in the number of intracellular electrons in a microbial fuel cell (MFC) helps to improve the MFC performance.

Results: In this study, we accumulated excess electrons intracellularly by knocking out the gene related to intracellular electron consumption in Saccharomyces cerevisiae, and the elevated intracellular electron pool positively influenced the performances of MFCs in terms of electricity production, while helping to increase ethanol production and achieve ethanol and electricity co-production, which in turn improved the utilization of substrates. The final knockout strain reached a maximum ethanol yield of 7.71 g/L and a maximum power density of 240 mW/m² in the MFC, which was 12 times higher than that of the control bacteria, with a 17.3% increase in energy utilization.

Conclusions: The knockdown of intracellular electron-consuming genes reported here allowed the accumulation of excess electrons in cells, and the elevated intracellular electron pool positively influenced the electrical production performance of the MFC. Furthermore, by knocking out the intracellular metabolic pathway, the yield of ethanol could be increased, and co-production of ethanol and electricity could be achieved. Thus, the MFC improved the utilization of the substrate.

Background: Increasing seed oil content (SOC) of Brassica napus has become one of the main plant breeding goals over the past decades. Lysophosphatidic acid acyltransferase (LPAT) performs an important molecular function by regulating the production of phosphatidic acid (PA), a key intermediate in the synthesis of membrane and storage lipids. However, the mechanism underlying the effect of LPAT on the SOC of B. napus remains unclear.

Results: In the present study, significant elevation of SOC was achieved by overexpressing BnLPAT2 and BnLPAT5 in B. napus. RNAi and CRISPR-Cas9 were also successfully used to knock down and knock out these two genes in B. napus where SOC significantly decreased. Meanwhile, we found an accumulation of lipid droplets and oil bodies in seeds of BnLPAT2 and BnLPAT5 overexpression lines, whereas an increase of sugar and protein in Bnlpat2 and Bnlpat5 mutant seeds. Sequential transcriptome analysis was further performed on the developing seeds of the BnLPAT2 and BnLPAT5 overexpression, knockdown, and knockout rapeseed lines. Most differentially expressed genes (DEGs) that were expressed in the middle and late stages of seed development were enriched in photosynthesis and lipid metabolism, respectively. The DEGs involved in fatty acid and lipid biosynthesis were active in the overexpression lines but were relatively inactive in the knockdown and knockout lines. Further analysis revealed that the biological pathways related to fatty acid/lipid anabolism and carbohydrate metabolism were specifically enriched in the BnLPAT2 overexpression lines.

Conclusions: BnLPAT2 and BnLPAT5 are essential for seed oil accumulation. BnLPAT2 preferentially promoted diacylglycerol synthesis to increase SOC, whereas BnLPAT5 tended to boost PA synthesis for membrane lipid generation. Taken together, BnLPAT2 and BnLPAT5 can jointly but differently promote seed oil accumulation in B. napus. This study provides new insights into the potential mechanisms governing the promotion of SOC by BnLPAT2 and BnLPAT5 in the seeds of B. napus.

Background: Purine nucleosides play essential roles in cellular physiological processes and have a wide range of applications in the fields of antitumor/antiviral drugs and food. However, microbial overproduction of purine nucleosides by de novo metabolic engineering remains a great challenge due to their strict and complex regulatory machinery involved in biosynthetic pathways.

Results: In this study, we designed an in silico-guided strategy for overproducing purine nucleosides based on a genome-scale metabolic network model in Bacillus subtilis. The metabolic flux was analyzed to predict two key backflow nodes, Drm (purine nucleotides toward PPP) and YwjH (PPP-EMP), to resolve the competitive relationship between biomass and purine nucleotide synthesis. In terms of the purine synthesis pathway, the first backflow node Drm was inactivated to block the degradation of purine nucleotides, which greatly increased the inosine production to 13.98-14.47 g/L without affecting cell growth. Furthermore, releasing feedback inhibition of the purine operon by promoter replacement enhanced the accumulation of purine nucleotides. In terms of the central carbon metabolic pathways, the deletion of the second backflow node YwjH and overexpression of Zwf were combined to increase inosine production to 22.01 ± 1.18 g/L by enhancing the metabolic flow of PPP. By switching on the flux node of the glucose-6-phosphate to PPP or EMP, the final inosine engineered strain produced up to 25.81 ± 1.23 g/L inosine by a pgi-based metabolic switch with a yield of 0.126 mol/mol glucose, a productivity of 0.358 g/L/h and a synthesis rate of 0.088 mmol/gDW/h, representing the highest yield in de novo engineered inosine bacteria. Under the guidance of this in silico-designed strategy, a general chassis bacterium was generated, for the first time, to efficiently synthesize inosine, adenosine, guanosine, IMP and GMP, which provides sufficient precursors for the synthesis of various purine intermediates.

Conclusions: Our study reveals that in silico-guided metabolic engineering successfully optimized the purine synthesis pathway by exploring efficient targets, which could be applied as a superior strategy for efficient biosynthesis of biotechnological products.

Background: Paenibacillus polymyxa is a typical plant growth-promoting rhizobacterium (PGPR), and synthesis of indole-3-acetic acid (IAA) is one of the reasons for its growth-promoting capacity. The synthetic pathways of IAA in P. polymyxa must be identified and modified.

Results: P. polymyxa SC2 and its spontaneous mutant SC2-M1 could promote plant growth by directly secreting IAA. Through metabonomic and genomic analysis, the genes patA, ilvB3, and fusE in the native IPyA pathway of IAA synthesis in strain SC2-M1 were predicted. A novel strong promoter P₀₄₄₂₀ was rationally selected, synthetically analyzed, and then evaluated on its ability to express IAA synthetic genes. Co-expression of three genes, patA, ilvB3, and fusE, increased IAA yield by 60% in strain SC2-M1. Furthermore, the heterogeneous gene iaam of the IAM pathway and two heterogeneous IPyA pathways of IAA synthesis were selected to improve the IAA yield of strain SC2-M1. The genes ELJP6_14505, ipdC, and ELJP6_00725 of the entire IPyA pathway from Enterobacter ludwigii JP6 were expressed well by promoter P₀₄₄₂₀ in strain SC2-M1 and increased IAA yield in the engineered strain SC2-M1 from 13 to 31 μg/mL, which was an increase of 138%.

Conclusions: The results of our study help reveal and enhance the IAA synthesis pathways of P. polymyxa and its future application.

Oxo chemicals are valuable chemicals for synthesizing a wide array of industrial and consumer products. However, producing of oxo chemicals is predominately through the chemical process called hydroformylation, which requires petroleum-sourced materials and generates abundant greenhouse gas. Current concerns on global climate change have renewed the interest in reducing greenhouse gas emissions and recycling the plentiful greenhouse gas. A carbon-neutral manner in this regard is producing oxo chemicals biotechnologically using greenhouse gas as C₁ feedstocks. Exemplifying isobutyraldehyde, this review demonstrates the significance of using greenhouse gas for oxo chemicals production. We highlight the current state and the potential of isobutyraldehyde synthesis with a special focus on the in vivo and in vitro scheme of C₁-based biomanufacturing. Specifically, perspectives and scenarios toward carbon- and nitrogen-neutral isobutyraldehyde production are proposed. In addition, key challenges and promising approaches for enhancing isobutyraldehyde bioproduction are thoroughly discussed. This study will serve as a reference case in exploring the biotechnological potential and advancing oxo chemicals production derived from C₁ feedstocks.

Background: Limonene is a cyclic monoterpene that has applications in the food, cosmetic, and pharmaceutical industries. The industrial production of limonene and its derivatives through plant extraction presents important drawbacks such as seasonal and climate issues, feedstock limitations, low efficiency and environmental concerns. Consequently, the implementation of efficient and eco-friendly bioprocesses for the production of limonene and other terpenes constitutes an attractive goal for microbial biotechnology. In this context, novel biocatalysts with the ability to produce limonene from alternative carbon sources will help to meet the industrial demands of limonene.

Results: Engineered strains of the industrial fungus Ashbya gossypii have been developed to produce limonene from xylose. The limonene synthase (LS) from Citrus limon was initially overexpressed together with the native HMG1 gene (coding for HMG-CoA reductase) to establish a limonene-producing platform from a xylose-utilizing A. gossypii strain. In addition, several strategies were designed to increase the production of limonene. Hence, the effect of mutant alleles of ERG20 (erg20^F95W and erg20^F126W) were evaluated together with a synthetic orthogonal pathway using a heterologous neryl diphosphate synthase. The lethality of the A. gossypii double mutant erg20^F95W-F126W highlights the indispensability of farnesyl diphosphate for the synthesis of essential sterols. In addition, the utilization of the orthogonal pathway, bypassing the Erg20 activity through neryl diphosphate, triggered a substantial increase in limonene titer (33.6 mg/L), without critically altering the fitness of the engineered strain. Finally, the overexpression of the native ERG12 gene further enhanced limonene production, which reached 336.4 mg/L after 96 h in flask cultures using xylose as the carbon source.

Conclusions: The microbial production of limonene can be carried out using engineered strains of A. gossypii from xylose-based carbon sources. The utilization of a synthetic orthogonal pathway together with the overexpression of ERG12 is a highly beneficial strategy for the production of limonene in A. gossypii. The strains presented in this work constitute a proof of principle for the production of limonene and other terpenes from agro-industrial wastes such as xylose-rich hydrolysates in A. gossypii.

Background: Lignocellulose is an important raw material for biomass-to-energy conversion, and it exhibits a complex but inefficient degradation mechanism. Microbial degradation is promising due to its environmental adaptability and biochemical versatility, but the pathways used by microbes for lignin degradation have not been fully studied. Degradation intermediates and complex metabolic pathways require more study.

Results: A novel actinomycete DF3-3, with the potential for lignin degradation, was screened and isolated. After morphological and molecular identification, DF3-3 was determined to be Streptomyces thermocarboxydus. The degradation of alkali lignin reached 31% within 15 days. Manganese peroxidase and laccase demonstrated their greatest activity levels, 1821.66 UL^-1 and 1265.58 UL^-1, respectively, on the sixth day. The highest lignin peroxidase activity was 480.33 UL^-1 on the fourth day. A total of 19 lignin degradation intermediates were identified by gas chromatography-mass spectrometry (GC-MS), including 9 aromatic compounds. Genome sequencing and annotation identified 107 lignin-degrading enzyme-coding genes containing three core enzymatic systems for lignin depolymerization: laccases, peroxidases and manganese peroxidase. In total, 7 lignin metabolic pathways were predicted.

Conclusions: Streptomyces thermocarboxydus strain DF3-3 has good lignin degradation ability. Degradation products and genomics analyses of DF3-3 show that it has a relatively complete lignin degradation pathway, including the β-ketoadipate pathway and peripheral reactions, gentisate pathway, anthranilate pathway, homogentisic pathway, and catabolic pathway for resorcinol. Two other pathways, the phenylacetate-CoA pathway and the 2,3-dihydroxyphenylpropionic acid pathway, are predicted based on genome data alone. This study provides the basis for future characterization of potential biotransformation enzyme systems for biomass energy conversion.

Background: Astaxanthin is a highly valuable ketocarotenoid with strong antioxidative activity and is natively accumulated upon environmental stress exposure in selected microorganisms. Green microalgae are photosynthetic, unicellular organisms cultivated in artificial systems to produce biomass and industrially relevant bioproducts. While light is required for photosynthesis, fueling carbon fixation processes, application of high irradiance causes photoinhibition and limits biomass productivity.

Results: Here, we demonstrate that engineered astaxanthin accumulation in the green alga Chlamydomonas reinhardtii conferred high light tolerance, reduced photoinhibition and improved biomass productivity at high irradiances, likely due to strong antioxidant properties of constitutively accumulating astaxanthin. In competitive co-cultivation experiments, astaxanthin-rich Chlamydomonas reinhardtii outcompeted its corresponding parental background strain and even the fast-growing green alga Chlorella vulgaris.

Conclusions: Metabolic engineering inducing astaxanthin and ketocarotenoids accumulation caused improved high light tolerance and increased biomass productivity in the model species for microalgae Chlamydomonas reinhardtii. Thus, engineering microalgal pigment composition represents a powerful strategy to improve biomass productivities in customized photobioreactors setups. Moreover, engineered astaxanthin accumulation in selected strains could be proposed as a novel strategy to outperform growth of other competing microalgal strains.

Background: Endoglucanases from thermophilic microorganisms are a valuable resource as they can be used in a wide variety of biotechnological applications including the valorisation of biomass and the production of biofuels. In the present work we analysed the metagenome from the hot spring Muiño da Veiga, located in the northwest of Spain (in the Galicia region), in search for novel thermostable endoglucanases.

Results: Sequence analysis of the metagenome revealed a promising enzyme (Cel776). Predictions on protein structure and conserved amino acid sequences were conducted, as well as expression in heterologous systems with Escherichia coli and Saccharomyces cerevisiae as the host. Cel776Ec was correctly expressed and purified by taking advantage of the His-Tag system, with a yield of 0.346 U/mL in the eluted fraction. Cel776Sc was expressed extracellulary and was easily recovered from the supernatant without the need of further purification, requiring only a concentration step by ultrafiltration, with a significantly higher yield of 531.95 U/mL, revealing a much more suitable system for production of large amounts of the enzyme. Their biochemical characterization revealed biotechnologically interesting enzymes. Both Cel776Ec and Cel776Sc had an optimal temperature of 80 °C and optimal pH of 5. Cel776Ec exhibited high thermostability maintaining its activity for 24 h at 60 °C and maintained its activity longer than Cel776Sc at increasing incubation temperatures. Moreover, its substrate specificity allowed the degradation of both cellulose and xylan. Whereas Cel776Ec was more active in the presence of calcium and magnesium, manganese was found to increase Cel776Sc activity. A stronger inhibitory effect was found for Cel776Ec than Cel776Sc adding detergent SDS to the reaction mix, whereas EDTA only significantly affected Cel776Sc activity.

Conclusions: Our study reports the discovery of a new promising biocatalyst for its application in processes, such as the production of biofuel and the saccharification of plant biomass, due to its bifunctional enzymatic activity as an endoglucanase and as a xylanase, as well as highlights the advantages of a yeast expression system over bacteria.

Background: Microalgal starch can be exploited for bioenergy, food, and bioplastics. Production of starch by green algae has been concerned for many years. Currently commonly used methods such as nutrient stress will affect cell growth, thereby inhibiting the production efficiency and quality of starch production. Simpler and more efficient control strategies need to be developed.

Result: We proposed a novel regulation method to promote the growth and starch accumulation by a newly isolated Chlorophyta Platymonas helgolandica. By adding exogenous glucose and controlling the appropriate circadian light and dark time, the highest dry weight accumulation 6.53 g L^-1 (Light:Dark = 12:12) can be achieved, and the highest starch concentration could reach 3.88 g L^-1 (Light:Dark = 6:18). The highest production rate was 0.40 g L^-1 d^-1 after 9 days of production. And this method helps to improve the ability to produce amylose, with the highest accumulation of 39.79% DW amylose. We also discussed the possible mechanism of this phenomenon through revealing changes in the mRNA levels of key genes.

Conclusion: This study provides a new idea to regulate the production of amylose by green algae. For the first time, it is proposed to combine organic carbon source addition and circadian rhythm regulation to increase the starch production from marine green alga. A new starch-producing microalga has been isolated that can efficiently utilize organic matter and grow with or without photosynthesis.