Biotechnology for Biofuels and Bioproducts最新文献

Background: Chlorophyll is a very important pigment involved in photosynthesis, while plant acyl-CoA biosynthesis is derived from plastid-localized fatty acids (FAs). Until now, the regulation of the acyl-CoA pathway for chlorophyll biosynthesis is still unknown.

Results: Here, we identified a long-chain acyl-CoA synthetase (LACS) gene BnLACS9 from Brassica napus. BnLACS9 complemented a LACS-deficient yeast strain YB525, which indicated that BnLACS9 has the LACS function. BnLACS9 was localized in the chloroplast envelope membrane, while mainly expressed in young leaves and flowers. Overexpression of BnLACS9 in Nicotiana benthamiana resulted in an increase in total CoA and MGDG content. In B. napus with overexpression of BnLACS9, the number of chloroplast grana lamellae and the chlorophyll content, as well as the MGDG and DGDG contents, increased compared to wild type. The net photosynthetic rate, dry weight of the entire plant and oil content of seeds increased significantly, accompanied by an increase in chlorophyll content. Transcriptome analysis revealed that overexpression of BnLACS9 improved the pathway of acyl-CoA biosynthesis and further improved the enzymes in the glycolipid synthesis pathway, while acyl-CoA was the substrate for glycolipid synthesis. The increased glycolipids, especially MGDG and DGDG, accelerated the formation of the chloroplast grana lamellae, which increased the number of chloroplast thylakoid grana lamella and further lead to increased chlorophyll content.

Conclusions: In the present study, we demonstrated that BnLACS9 played a crucial role in glycolipids and chlorophyll biosynthesis in B. napus. The results also provide a new direction and theoretical basis for the improvement of the agronomic traits of plants.

Background: Previous studies have revealed that some Auxiliary Activity family 9 (AA9) lytic polysaccharide monooxygenases (LPMOs) oxidize and degrade certain types of xylans when incubated with mixtures of xylan and cellulose. Here, we demonstrate that the xylanolytic activities of two xylan-active LPMOs, TtLPMO9E and TtLPMO9G from Thermothielavioides terrestris, strongly depend on the presence of xylan substitutions.

Results: Using mixtures of phosphoric acid-swollen cellulose (PASC) and wheat arabinoxylan (WAX), we show that removal of arabinosyl substitutions with a GH62 arabinofuranosidase resulted in better adsorption of xylan to cellulose, and enabled LPMO-catalyzed cleavage of this xylan. Furthermore, experiments with mixtures of PASC and arabinoglucuronoxylan from spruce showed that debranching of xylan with the GH62 arabinofuranosidase and a GH115 glucuronidase promoted LPMO activity. Analyses of mixtures with PASC and (non-arabinosylated) beechwood glucuronoxylan showed that GH115 action promoted LPMO activity also on this xylan. Remarkably, when WAX was incubated with Avicel instead of PASC in the presence of the GH62, both xylan and cellulose degradation by the LPMO9 were impaired, showing that the formation of cellulose-xylan complexes and their susceptibility to LPMO action also depend on the properties of the cellulose. These debranching effects not only relate to modulation of the cellulose-xylan interaction, which influences the conformation and rigidity of the xylan, but likely also affect the LPMO-xylan interaction, because debranching changes the architecture of the xylan surface.

Conclusions: Our results shed new light on xylanolytic LPMO9 activity and on the functional interplay and possible synergies between the members of complex lignocellulolytic enzyme cocktails. These findings will be relevant for the development of future lignocellulolytic cocktails and biomaterials.

Succinic acid (SA) is an intermediate product of the tricarboxylic acid cycle (TCA) and is one of the most significant platform chemicals for the production of various derivatives with high added value. Due to the depletion of fossil raw materials and the demand for eco-friendly energy sources, SA biosynthesis from renewable energy sources is gaining attention for its environmental friendliness. This review comprehensively analyzes strategies for the bioconversion of lignocellulose to SA based on the lignocellulose pretreatment processes and cellulose hydrolysis and fermentation principles and highlights the research progress on acid production and SA utilization under different microbial culture conditions. In addition, the fermentation efficiency of different microbial strains for the production of SA and the main challenges were analyzed. The future application directions of SA derivatives were pointed out. It is expected that this research will provide a reference for the optimization of SA production from lignocellulose.

Background: β-Farnesene is a sesquiterpene with versatile industrial applications. The production of β-farnesene from waste lipid feedstock is an attractive method for sustainable production and recycling waste oil. Yarrowia lipolytica is an unconventional oleaginous yeast, which can use lipid feedstock and has great potential to synthesize acetyl-CoA-derived chemicals.

Results: In this study, we engineered Y. lipolytica to produce β-farnesene from lipid feedstock. To direct the flux of acetyl-CoA, which is generated from lipid β-oxidation, to β-farnesene synthesis, the mevalonate synthesis pathway was compartmentalized into peroxisomes. β-Farnesene production was then engineered by the protein engineering of β-farnesene synthase and pathway engineering. The regulation of lipid metabolism by enhancing β-oxidation and eliminating intracellular lipid synthesis was further performed to improve the β-farnesene synthesis. As a result, the final β-farnesene production with bio-engineering reached 35.2 g/L and 31.9 g/L using oleic acid and waste cooking oil, respectively, which are the highest β-farnesene titers reported in Y. lipolytica.

Conclusions: This study demonstrates that engineered Y. lipolytica could realize the sustainable production of value-added acetyl-CoA-derived chemicals from waste lipid feedstock.

Background: With the development of metabolic engineering and synthetic biology, the biosynthesis of aromatic compounds has attracted much attention. Cinnamylamine is an aromatic compound derived from L-phenylalanine, which is used in the synthesis of biologically active molecules, including drugs, and energetic materials. Cinnamylamine has been mainly synthesized by chemical methods to date, and few reports have focused on the biosynthesis of cinnamylamine. Therefore, it is desirable to establish an efficient biosynthesis method for cinnamylamine.

Results: The ω-aminotransferase Cv-ωTA from Chromobacterium violaceum has been demonstrated to have high enzyme activity in the conversion of cinnamaldehyde to cinnamylamine. To prevent the preferable conversion of cinnamaldehyde to cinnamyl alcohol in wild-type Escherichia coli, the E. coli MG1655 strain with reduced aromatic aldehyde reduction (RARE) in which six aldehyde ketone reductase and alcohol dehydrogenase genes have been knocked out was employed. Then, the carboxylic acid reductase from Neurospora crassa (NcCAR) and phosphopantetheinyl transferase (PPTase) from E. coli were screened for a high conversion rate of cinnamic acid to cinnamaldehyde. To shift the equilibrium of the reaction toward cinnamylamine, saturation mutagenesis of Cv-ωTA at key amino acid residues was performed, and Cv-ωTA Y168G had the highest conversion rate with 88.56 mg/L cinnamylamine obtained after 4 h of fermentation. Finally, by optimizing the substrates and the supply of the cofactors, PLP and NADPH, in the fermentation, the yield of cinnamylamine in engineered E. coli reached 523.15 mg/L.

Conclusion: We achieved the first biosynthesis of cinnamylamine using cinnamic acid as the precursor in E. coli using a combinatorial metabolic engineering strategy. This study provides a reference for the biosynthesis of other amine compounds and lays a foundation for the de novo synthesis of cinnamylamine.

Background: Based on known metabolic response to excess free fatty acid (FFA) products, cyanobacterium Synechocystis sp. PCC 6803 preferentially both recycles via FFA recycling process and secrets them into medium. Engineered cyanobacteria with well growth and highly secreted FFA capability are considered best resources for biofuel production and sustainable biotechnology. In this study, to achieve the higher FFA secretion goal, we successfully constructs Synechocystis sp. PCC 6803 mutants disrupting genes related to FFA recycling reaction (aas gene encoding acyl-acyl carrier protein synthetase), and surface layer protein (encoded by sll1951).

Results: Three Synechocystis sp. PCC 6803 engineered strains, including two single mutants lacking aas (KA) and sll1951 (KS), and one double mutant lacking both aas and sll1951 (KAS), significantly secreted FFAs higher than that of wild type (WT). Certain increase of secreted FFAs was noted when cells were exposed to nitrogen-deficient conditions, BG₁₁-half N and BG₁₁-N conditions, with the exception of strain KS. Under BG₁₁-N condition at day 10, strain KAS strikingly secreted FFAs products up to 40%w/DCW or 238.1 mg/L, with trace amounts of PHB. Unexpectedly, strain KS, with S-layer disruption, appeared to have endured longer in BG₁₁-N growth medium. This strain KS significantly acclimated to the BG₁₁-N environment by accumulating a greater glycogen pool with lower FFA production, whereas strain KA favored higher PHB and intracellular lipid accumulations with moderate FFA secretion.

Conclusions: Mutations of both aas and sll1951 genes in Synechocystis sp. PCC 6803 significantly improved the productivity of secreted FFAs, especially under nitrogen deprivation.

Background: L-Ornithine is an important medicinal intermediate that is mainly produced by microbial fermentation using glucose as the substrate. To avoid competition with human food resources, there is an urgent need to explore alternative carbon sources for L-ornithine production. In a previous study, we constructed an engineered strain, Corynebacterium glutamicum MTL13, which produces 54.56 g/L of L-ornithine from mannitol. However, compared with the titers produced using glucose as a substrate, the results are insufficient, and further improvement is required.

Results: In this study, comparative transcriptome profiling of MTL01 cultivated with glucose or mannitol was performed to identify novel targets for engineering L-ornithine-producing strains. Guided by the transcriptome profiling results, we modulated the expression of qsuR (encoding a LysR-type regulator QsuR), prpC (encoding 2-methylcitrate synthase PrpC), pdxR (encoding a MocR-type regulator PdxR), acnR (encoding a TetR-type transcriptional regulator AcnR), CGS9114_RS08985 (encoding a hypothetical protein), and CGS9114_RS09730 (encoding a TetR/AcrR family transcriptional regulator), thereby generating the engineered strain MTL25 that can produce L-ornithine at a titer of 93.6 g/L, representing a 71.6% increase as compared with the parent strain MTL13 and the highest L-ornithine titer reported so far for C. glutamicum.

Conclusions: This study provides novel indirect genetic targets for enhancing L-ornithine accumulation on mannitol and lays a solid foundation for the biosynthesis of L-ornithine from marine macroalgae, which is farmed globally as a promising alternative feedstock.

Background: The demand for naturally derived products is continuously growing. Nutraceuticals such as pre- and post-biotics, antioxidants and vitamins are prominent examples in this scenario, but many of them are mainly produced by chemical synthesis. The global folate market is expected to register a CAGR of 5.3% from 2019 to 2024 and reach USD 1.02 billion by the end of 2024. Vitamin B₉, commonly known as folate, is an essential micronutrient for humans. Acting as a cofactor in one-carbon transfer reactions, it is involved in many biochemical pathways, among which the synthesis of nucleotides and amino acids. In addition to plants, many microorganisms can naturally produce it, and this can pave the way for establishing production processes. In this work, we explored the use of Scheffersomyces stipitis for the production of natural vitamin B₉ by microbial fermentation as a sustainable alternative to chemical synthesis.

Results: Glucose and xylose are the main sugars released during the pretreatment and hydrolysis processes of several residual lignocellulosic biomasses (such as corn stover, wheat straw or bagasse). We optimized the growth conditions in minimal medium formulated with these sugars and investigated the key role of oxygenation and nitrogen source on folate production. Vitamin B₉ production was first assessed in shake flasks and then in bioreactor, obtaining a folate production up to 3.7 ± 0.07 mg/L, which to date is the highest found in literature when considering wild type microorganisms. Moreover, the production of folate was almost entirely shifted toward reduced vitamers, which are those metabolically active for humans.

Conclusions: For the first time, the non-Saccharomyces yeast S. stipitis was used to produce folate. The results confirm its potential as a microbial cell factory for folate production, which can be also improved both by genetic engineering strategies and by fine-tuning the fermentation conditions and nutrient requirements.

Background: Anaerobic fungi of the phylum Neocallimastigomycota have a high biotechnological potential due to their robust lignocellulose degrading capabilities and the production of several valuable metabolites like hydrogen, acetate, formate, lactate, and ethanol. The metabolism of these fungi, however, remains poorly understood due to limitations of the current cultivation strategies in still-standing bottles, thereby restricting the comprehensive evaluation of cultivation conditions.

Results: We describe the analysis of growth conditions and their influence on the metabolism of the previously isolated fungus Neocallimastix cameroonii G341. We established a bioreactor process in a stirred tank, enabling cultivation under defined conditions. The optimal growth temperature for the fungus was between 38.5 °C and 41.5 °C, while the optimal pH was 6.6-6.8. Like other dark fermentation systems, hydrogen production is dependent on the hydrogen partial pressure and pH. Shaking the bottles or stirring the fermenters led to an increase in hydrogen and a decrease in lactate and ethanol production. Regulation of the pH to 6.8 in the fermenter nearly doubled the amount of produced hydrogen.

Conclusions: Novel insights into the metabolism of Neocallimastix cameroonii were gained, with hydrogen being the preferred way of electron disposal over lactate and ethanol. In addition, our study highlights the potential application of the fungus for hydrogen production from un-pretreated biomass. Finally, we established the first cultivation of an anaerobic fungus in a stirred tank reactor system.

Background: Microalgal lipid production has attracted global attention in next-generation biofuel research. Nitrogen starvation, which drastically suppresses cell growth, is a common and strong trigger for lipid accumulation in microalgae. We previously developed a mutant Chlamydomonas sp. KAC1801, which can accumulate lipids irrespective of the presence or absence of nitrates. This study aimed to develop a feasible strategy for stable and continuous lipid production through semi-continuous culture of KAC1801.

Results: KAC1801 continuously accumulated > 20% lipid throughout the subculture (five generations) when inoculated with a dry cell weight of 0.8-0.9 g L^-1 and cultured in a medium containing 18.7 mM nitrate, whereas the parent strain KOR1 accumulated only 9% lipid. Under these conditions, KAC1801 continuously produced biomass and consumed nitrates. Lipid productivity of 116.9 mg L^-1 day^-1 was achieved by semi-continuous cultivation of KAC1801, which was 2.3-fold higher than that of KOR1 (50.5 mg L^-1 day^-1). Metabolome and transcriptome analyses revealed a depression in photosynthesis and activation of nitrogen assimilation in KAC1801, which are the typical phenotypes of microalgae under nitrogen starvation.

Conclusions: By optimizing nitrate supply and cell density, a one-step cultivation system for Chlamydomonas sp. KAC1801 under nitrate-replete conditions was successfully developed. KAC1801 achieved a lipid productivity comparable to previously reported levels under nitrogen-limiting conditions. In the culture system of this study, metabolome and transcriptome analyses revealed a nitrogen starvation-like response in KAC1801.