Biotechnology for Biofuels and Bioproducts最新文献

Background: With D-xylose being the second most abundant sugar in nature, its conversion into products could significantly improve biomass-based process economy. There are two well-studied phosphorylative pathways for D-xylose metabolism. One is isomerase pathway mainly found in bacteria, and the other one is oxo-reductive pathway that always exists in fungi. Except for these two pathways, there are also non-phosphorylative pathways named xylose oxidative pathways and they have several advantages over traditional phosphorylative pathways. In Myceliophthora thermophila, D-xylose can be metabolized through oxo-reductive pathway after plant biomass degradation. The survey of non-phosphorylative pathways in this filamentous fungus will offer a potential way for carbon-efficient production of fuels and chemicals using D-xylose.

Results: In this study, an alternative for utilization of D-xylose, the non-phosphorylative Weimberg pathway was established in M. thermophila. Growth on D-xylose of strains whose D-xylose reductase gene was disrupted, was restored after overexpression of the entire Weimberg pathway. During the construction, a native D-xylose dehydrogenase with highest activity in M. thermophila was discovered. Here, M. thermophila was also engineered to produce 1,2,4-butanetriol using D-xylose through non-phosphorylative pathway. Afterwards, transcriptome analysis revealed that the D-xylose dehydrogenase gene was obviously upregulated after deletion of D-xylose reductase gene when cultured in a D-xylose medium. Besides, genes involved in growth were enriched in strains containing the Weimberg pathway.

Conclusions: The Weimberg pathway was established in M. thermophila to support its growth with D-xylose being the sole carbon source. Besides, M. thermophila was engineered to produce 1,2,4-butanetriol using D-xylose through non-phosphorylative pathway. To our knowledge, this is the first report of non-phosphorylative pathway recombinant in filamentous fungi, which shows great potential to convert D-xylose to valuable chemicals.

Background: Pyrroloquinoline quinone (PQQ), a cofactor for bacterial dehydrogenases, is associated with biological processes such as mitochondriogenesis, reproduction, growth, and aging. Due to the extremely high cost of chemical synthesis and low yield of microbial synthesis, the election of effective strains and the development of dynamic fermentation strategies for enhancing PQQ production are meaningful movements to meet the large-scale industrial requirements.

Results: A high-titer PQQ-producing mutant strain, Hyphomicrobium denitrificans FJNU-A26, was obtained by integrating ARTP (atmospheric and room‑temperature plasma) mutagenesis, adaptive laboratory evolution and high-throughput screening strategies. Afterward, the systematic optimization of the fermentation medium was conducted using a one-factor-at-a-time strategy and response surface methodology to increase the PQQ concentration from 1.02 to 1.37 g/L. The transcriptional analysis using qRT-PCR revealed that the expression of genes involved in PQQ biosynthesis were significantly upregulated when the ARTP-ALE-derived mutant was applied. Furthermore, a novel two-stage pH control strategy was introduced to address the inconsistent effects of the pH value on cell growth and PQQ production. These combined strategies led to a 148% increase in the PQQ concentration compared with that of the initial strain FJNU-6, reaching 1.52 g/L with a yield of 40.3 mg/g DCW after 144 h of fed-batch fermentation in a 5-L fermenter.

Conclusion: The characteristics above suggest that FJNU-A26 represents an effective candidate as an industrial PQQ producer, and the integrated strategies can be readily extended to other microorganisms for the large-scale production of PQQ.

Background: Microalgae are promising feedstocks for production of renewable biofuels and value-added bioproducts. Temperature and nitrogen supply are important environmental and nutritional factors affecting the growth and metabolism of microalgae, respectively. In this study, the growth and lipid accumulation of filamentous microalgae Xanthonema hormidioides under different temperatures (5, 7, 10, 15, 20, 25, 27 and 30 °C) and initial nitrogen concentrations (3, 9, 18 mM) were investigated, and its adaptive mechanisms of tolerance to low temperature and nitrogen stress were analysis by proteomics.

Results: The optimum temperature range for the growth of X. hormidioides was between 15 and 20 °C, and the algal cells had slow growth rate at 5 °C and could not survive at 30 °C. The maximum biomass concentration was 11.73 g L^-1 under the temperature of 20 °C, and the highest total lipid content was 56.63% of dry weight. Low temperature did not change the fatty acids profiles but promoted the accumulation of unsaturated fatty acids of X. hormidioides. The maximum contents of palmitoleic acid, eicosapentaenoic acid and total fatty acid were 23.64%, 2.49% and 41.14% of dry weight, respectively. Proteomics was performed under three temperature (7, 15, 25 °C), two nitrogen concentrations (3 and 18 mM) and two cultivation times (day 3 and 12). A total of 6503 proteins were identified. In the low temperature, photosynthesis-related proteins were down-regulated to protect the photosynthetic apparatus. The up-regulation of key enzymes DGAT and PDAT demonstrated the accumulation of TAGs under low nitrogen treatment. The proteins related to ribosome, phosphatidylinositol signaling system, antioxidant system and cold shock proteins (CSPs) in X. hormidioides were co-upregulated under the treatment of low temperature, which can alleviate the damages induced by temperature stress and maintain the normal growth and metabolism of algal cells.

Conclusions: X. hormidioides is a psychrotolerant microalga. It is an oleaginous filamentous microalga containing hyper palmitoleic acid and a certain amount of eicosapentaenoic acid with great potential for biofuel development, as well as for applications in nutritional health products and other industries.

Background: Lignocellulose is a valuable carbon source for the production of biofuels and biochemicals, thus having the potential to substitute fossil resources. Consolidated bio-saccharification (CBS) is a whole-cell-based catalytic technology previously developed to produce fermentable sugars from lignocellulosic agricultural wastes. The deep-sea yeast strain Rhodotorula paludigena P4R5 can produce extracellular polyol esters of fatty acids (PEFA) and intracellular single-cell oils (SCO) simultaneously. Therefore, the integration of CBS and P4R5 fermentation processes would achieve high-value-added conversion of lignocellulosic biomass.

Results: The strain P4R5 could co-utilize glucose and xylose, the main monosaccharides from lignocellulose, and also use fructose and arabinose for PEFA and SCO production at high levels. By regulating the sugar metabolism pathways for different monosaccharides, the strain could produce PEFA with a single type of polyol head. The potential use of PEFA as functional micelles was also determined. Most importantly, when sugar-rich CBS hydrolysates derived from corn stover or corncob residues were used to replace grain-derived pure sugars for P4R5 fermentation, similar PEFA and SCO productions were obtained, indicating the robust conversion of non-food corn plant wastes to high-value-added glycolipids and lipids. Since the produced PEFA could be easily collected from the culture via short-time standing, we further developed a semi-continuous process for PEFA production from corncob residue-derived CBS hydrolysate, and the PEFA titer and productivity were enhanced up to 41.1 g/L and 8.22 g/L/day, respectively.

Conclusions: Here, we integrated the CBS process and the P4R5 fermentation for the robust production of high-value-added PEFA and SCO from non-food corn plant wastes. Therefore, this study suggests a feasible way for lignocellulosic agro-waste utilization and the potential application of P4R5 in industrial PEFA production.

Background: Methanol, a promising non-food fermentation substrate, has gained increasing interest as an alternative feedstock to sugars for the bio-based production of value-added chemicals. Butyribacterium methylotrophicum, one of methylotrophic-acetogenic bacterium, is a promising host to assimilate methanol coupled with CO₂ fixation for the production of organic acids, such as butyric acid. Although the methanol utilization pathway has been identified in B. methylotrophicum, little knowledge was currently known about its regulatory targets, limiting the rational engineering to improve methanol utilization.

Results: In this study, we found that methanol assimilation of B. methylotrophicum could be significantly improved when using corn steep liquor (CSL) as the co-substrate. The further investigation revealed that high level of lysine was responsible for enhanced methanol utilization. Through the transcriptome analysis, we proposed a potential mechanism by which lysine confers improved methylotrophy via modulating NikABCDE and FhuBCD transporters, both of which are involved in the uptake of cofactors essential for enzymes of methanol assimilation. The improved methylotrophy was also confirmed by overexpressing NikABCDE or FhuBCD operon. Finally, the de novo synthetic pathway of lysine was further engineered and the methanol utilization and butyric acid production of B. methylotrophicum were improved by 63.2% and 79.7%, respectively. After an optimization of cultivation medium, 3.69 g/L of butyric acid was finally achieved from methanol with a yield of 76.3%, the highest level reported to date.

Conclusion: This study revealed a novel mechanism to regulate methanol assimilation by lysine in B. methylotrophicum and engineered it to improve methanol bioconversion to butyric acid, culminating in the synthesis of the highest butyric acid titer reported so far in B. methylotrophicum. What's more, our work represents a further advancement in the engineering of methylotrophic-acetogenic bacterium to improve C1-compound utilization.

Background: (R)-mandelic acid (R-MA) is a highly valuable hydroxyl acid in the pharmaceutical industry. However, biosynthesis of optically pure R-MA remains significant challenges, including the lack of suitable catalysts and high toxicity to host strains. Adaptive laboratory evolution (ALE) was a promising and powerful strategy to obtain specially evolved strains.

Results: Herein, we report a new cell factory of the Gluconobacter oxydans to biocatalytic styrene oxide into R-MA by utilizing the G. oxydans endogenous efficiently incomplete oxidization and the epoxide hydrolase (SpEH) heterologous expressed in G. oxydans. With a new screened strong endogenous promoter P₁₂₇₈₀, the production of R-MA was improved to 10.26 g/L compared to 7.36 g/L of using P_lac. As R-MA showed great inhibition for the reaction and toxicity to cell growth, adaptive laboratory evolution (ALE) strategy was introduced to improve the cellular R-MA tolerance. The adapted strain that can tolerate 6 g/L R-MA was isolated (named G. oxydans STA), while the wild-type strain cannot grow under this stress. The conversion rate was increased from 0.366 g/L/h of wild type to 0.703 g/L/h by the recombinant STA, and the final R-MA titer reached 14.06 g/L. Whole-genome sequencing revealed multiple gene-mutations in STA, in combination with transcriptome analysis under R-MA stress condition, we identified five critical genes that were associated with R-MA tolerance, among which AcrA overexpression could further improve R-MA titer to 15.70 g/L, the highest titer reported from bulk styrene oxide substrate.

Conclusions: The microbial engineering with systematic combination of static regulation, ALE, and transcriptome analysis strategy provides valuable solutions for high-efficient chemical biosynthesis, and our evolved G. oxydans would be better to serve as a chassis cell for hydroxyl acid production.

Background: Microtubules in cells are closely related to the growth and metabolism of microalgae. To date, the study of microalgal microtubules has mainly concentrated on revealing the relationship between microtubule depolymerization and synthesis of precursors for flagellar regeneration. While information on the link between microtubule depolymerization and biosynthesis of precursors for complex organic matter (such as lipid, carbohydrate and protein), is still lacking, a better understanding of this could help to achieve a breakthrough in lipid regulation. With the aim of testing the assumption that microtubule disruption could regulate carbon precursors and redirect carbon flow to promote lipid accumulation, Chlorella sorokiniana SDEC-18 was pretreated with different concentrations of oryzalin.

Results: Strikingly, microalgae that were pretreated with 1.5 mM oryzalin accumulated lipid contents of 41.06%, which was attributed to carbon redistribution induced by microtubule destruction. To promote the growth of microalgae, two-stage cultivation involving microtubule destruction was employed, which resulted in the lipid productivity being 1.44 times higher than that for microalgae with routine single-stage cultivation, as well as yielding a desirable biodiesel quality following from increases in monounsaturated fatty acid (MUFA) content. Furthermore, full extraction of lipid was achieved after only a single extraction step, because microtubule destruction caused removal of cellulose synthase and thereby blocked cellulose biosynthesis.

Conclusions: This study provides an important advance towards observation of microtubules in microalgae through immunocolloidal gold techniques combined with TEM. Moreover, the observation of efficient lipid accumulation and increased cell fragility engendered by microtubule destruction has expanded our knowledge of metabolic regulation by microtubules. Finally, two-stage cultivation involving microtubule destruction has established ideal growth, coupling enhanced lipid accumulation and efficient oil extraction; thus gaining advances in both applied and fundamental research in algal biodiesel production.

Background: Bacillus subtilis is generally regarded as a ubiquitous facultative anaerobe. Oxygen is the major electron acceptor of B. subtilis, and when oxygen is absent, B. subtilis can donate electrons to nitrate or perform fermentation. An anode electrode can also be used by microorganisms as the electron sink in systems called anodic electro-fermentation. The facultative anaerobic character of B. subtilis makes it an excellent candidate to explore with different electron acceptors, such as an anode. This study aimed to optimise industrial aerobic bioprocesses using alternative electron acceptors. In particular, different end product spectrum of B. subtilis with various electron acceptors, including anode from the electro-fermentation system, was investigated.

Results: B. subtilis was grown using three electron acceptors, i.e. oxygen, nitrate and anode (poised at a potential of 0.7 V vs. standard hydrogen electrode). The results showed oxygen had a crucial role for cells to remain metabolically active. When nitrate or anode was applied as the sole electron acceptor anaerobically, immediate cell lysis and limited glucose consumption were observed. In anode-assisted electro-fermentation with a limited aeration rate, acetoin, as the main end product showed the highest yield of 0.78 ± 0.04 mol_product/mol_glucose, two-fold higher than without poised potential (0.39 ± 0.08 mol_product/mol_glucose).

Conclusions: Oxygen controls B. subtilis biomass growth, alternative electron acceptors utilisation and metabolites formation. Limited oxygen/air supply enabled the bacteria to donate excess electrons to nitrate or anode, leading to steered product spectrum. The anode-assisted electro-fermentation showed its potential to boost acetoin production for future industrial biotechnology applications.

Background: Lignin is an attractive alternative for producing biobased chemicals. It is the second major component of the plant cell wall and is an abundant natural source of aromatic compounds. Lignin degradation using microbial oxidative enzymes that depolymerize lignin and catabolize aromatic compounds into central metabolic intermediates is a promising strategy for lignin valorization. However, the intrinsic heterogeneity and recalcitrance of lignin severely hinder its biocatalytic conversion. In this context, examining microbial degradation systems can provide a fundamental understanding of the pathways and enzymes that are useful for lignin conversion into biotechnologically relevant compounds.

Results: Lignin-degrading catabolism of a novel Rhodosporidium fluviale strain LM-2 was characterized using multi-omic strategies. This strain was previously isolated from a ligninolytic microbial consortium and presents a set of enzymes related to lignin depolymerization and aromatic compound catabolism. Furthermore, two catabolic routes for producing 4-vinyl guaiacol and vanillin were identified in R. fluviale LM-2.

Conclusions: The multi-omic analysis of R. fluviale LM-2, the first for this species, elucidated a repertoire of genes, transcripts, and secreted proteins involved in lignin degradation. This study expands the understanding of ligninolytic metabolism in a non-conventional yeast, which has the potential for future genetic manipulation. Moreover, this work unveiled critical pathways and enzymes that can be exported to other systems, including model organisms, for lignin valorization.

Background: Cyanobacteria have emerged as highly efficient organisms for the production of chemicals and biofuels. Yet, the productivity of the cell has been low for commercial application. Cyanobacterial photobiotransformations utilize photosynthetic electrons to form reducing equivalents, such as NADPH-to-fuel biocatalytic reactions. These photobiotransformations are a measure to which extent photosynthetic electrons can be deviated toward heterologous biotechnological processes, such as the production of biofuels. By expressing oxidoreductases, such as YqjM from Bacillus subtilis in Synechocystis sp. PCC 6803, a high specific activity was obtained in the reduction of maleimides. Here, we investigated the possibility to accelerate the NAD(P)H-consuming redox reactions by addition of carbohydrates as exogenous carbon sources such as D-Glucose under light and darkness.

Results: A 1.7-fold increase of activity (150 µmol min^-1 g_DCW^-1) was observed upon addition of D-Glucose at an OD₇₅₀ = 2.5 (DCW = 0.6 g L^-1) in the biotransformation of 2-methylmaleimide. The stimulating effect of D-Glucose was also observed at higher cell densities in light and dark conditions as well as in the reduction of other substrates. No increase in both effective photosynthetic yields of Photosystem II and Photosystem I was found upon D-Glucose addition. However, we observed higher NAD(P)H fluorescence when D-Glucose was supplemented, suggesting increased glycolytic activity. Moreover, the system was scaled-up (working volume of 200 mL) in an internally illuminated Bubble Column Reactor exhibiting a 2.4-fold increase of specific activity under light-limited conditions.

Conclusions: Results show that under photoautotrophic conditions at a specific activity of 90 µmol min^-1 g_DCW^-1, the ene-reductase YqjM in Synechocystis sp. PCC 6803 is not NAD(P)H saturated, which is an indicator that an increase of the rates of heterologous electron consuming processes for catalysis and biofuel production will require funnelling further reducing power from the photosynthetic chain toward heterologous processes.