Editing Streptomyces genome using target AID system fused with UGI-degradation tag.

The utilization of Streptomyces as a microbial chassis for developing innovative drugs and medicinal compounds showcases its capability to produce bioactive natural substances. Recent focus on the clustered regularly interspaced short palindromic repeat (CRISPR) technology highlights its potential in genome editing. However, applying CRISPR technology in certain microbial strains, particularly Streptomyces, encounters specific challenges. These challenges include achieving efficient gene expression and maintaining genetic stability, which are critical for successful genome editing. To overcome these obstacles, an innovative approach has been developed that combines several key elements: activation-induced cytidine deaminase (AID), nuclease-deficient cas9 variants (dCas9), and Petromyzon marinus cytidine deaminase 1 (PmCDA1). In this study, this novel strategy was employed to engineer a Streptomyces coelicolor strain. The target gene was actVA-ORF4 (SCO5079), which is involved in actinorhodin production. The engineering process involved introducing a specific construct [pGM1190-dcas9-pmCDA-UGI-AAV-actVA-ORF4 (SCO5079)] to create a CrA10 mutant strain. The resulting CrA10 mutant strain did not produce actinorhodin. This outcome highlights the potential of this combined approach in the genetic manipulation of Streptomyces. The failure of the CrA10 mutant to produce actinorhodin conclusively demonstrates the success of gene editing at the targeted site, affirming the effectiveness of this method for precise genetic modifications in Streptomyces.

Complete sequence randomness of lactate-based copolymers (LAHBs) with varied lactate monomer fractions employing a series of propionyl-CoA transferases.

Previously, we biosynthesized an evolved version of a bio-based polylactide (PLA) on microbial platforms using our engineered lactate-polymerizing enzyme (LPE). This lactate (LA)-based copolyester, LAHB, has advantages over PLA, including improved flexibility and biodegradability, and its properties can be regulated through the LA fraction. To expand the LA-incorporation capacity and improve polymer properties, in the state of in vivo LAHB production, propionyl-CoA transferases (PCTs) that exhibited enhanced production of LA-CoA than the conventional PCTs were selected. Here, the present study has demonstrated that the LA fraction of LAHB could be altered using various PCTs. Enhanced PCT performance was achieved by balancing polymer production and cell growth. Both events are governed by the use of acetyl-CoA, a commonly shared key metabolite. This could be attributed to the different reactivities of individual PCTs towards acetyl-CoA, which serves both as a CoA donor and a leading compound in the TCA cycle. Interestingly, we found complete sequence randomness in the LAHB copolymers, independent of the LA fraction. The mechanism of LA fraction-independent sequence randomness is discussed. This new PCT-based strategy synergistically combines with the evolution of LPE to advance the LAHB project, and enables us to perform advanced applications other than LAHB production utilizing CoA-linked substrates.

Production of single cell oil by Lipomyces starkeyi from waste plant oil generated by the palm oil mill industry.

Only a few reports available about the assimilation of hydrophobic or oil-based feedstock as carbon sources by Lipomyces starkeyi. In this study, the ability of L. starkeyi to efficiently utilize free fatty acids (FFAs) and real biomass like palm acid oil (PAO) as well as crude palm kernel oil (CPKO) for growth and lipid production was investigated. PAO, CPKO, and FFAs were evaluated as sole carbon sources or in the mixed medium containing glucose. L. starkeyi was able to grow on the medium supplemented with PAO and FFAs, which contained long-chain length FAs and accumulated lipids up to 35% (w/w) of its dry cell weight. The highest lipid content and lipid concentration were achieved at 50% (w/w) and 10.1 g/L, respectively, when L. starkeyi was cultured in nitrogen-limited mineral medium (-NMM) supplemented with PAO emulsion. Hydrophobic substrate like PAO could be served as promising carbon source for L. starkeyi.

Comparative responses of flocculating and nonflocculating yeasts to cell density and chemical stress in lactic acid fermentation.

While flocculation has demonstrated its efficacy in enhancing yeast robustness and ethanol production, its potential application for lactic acid fermentation remains largely unexplored. Our study examined the differences between flocculating and nonflocculating Saccharomyces cerevisiae strains in terms of their metabolic dynamics when incorporating an exogenous lactic acid pathway, across varying cell densities and in the presence of lignocellulose-derived byproducts. Comparative gene expression profiles revealed that cultivating a nonflocculant strain at higher cell density yielded a substantial upregulation of genes associated with glycolysis, energy metabolism, and other key pathways, resulting in elevated levels of fermentation products. Meanwhile, the flocculating strain displayed an inherent ability to sustain high glycolytic activity regardless of the cell density. Moreover, our investigation revealed a significant reduction in glycolytic activity under chemical stress, potentially attributable to diminished ATP supply during the energy investment phase. Conversely, the formation of flocs in the flocculating strain conferred protection against toxic chemicals present in the medium, fostering more stable lactic acid production levels. Additionally, the distinct flocculation traits observed between the two examined strains may be attributed to variations in the nucleotide sequences of the flocculin genes and their regulators. This study uncovers the potential of flocculation for enhanced lactic acid production in yeast, offering insights into metabolic mechanisms and potential gene targets for strain improvement.

Sucrose Signaling Contributes to the Maintenance of Vascular Cambium by Inhibiting Cell Differentiation.

Plants produce sugars by photosynthesis and use them for growth and development. Sugars are transported from source-to-sink organs via the phloem in the vasculature. It is well known that vascular development is precisely controlled by plant hormones and peptide hormones. However, the role of sugars in the regulation of vascular development is poorly understood. In this study, we examined the effects of sugars on vascular cell differentiation using a vascular cell induction system named 'Vascular Cell Induction Culture System Using Arabidopsis Leaves' (VISUAL). We found that sucrose has the strongest inhibitory effect on xylem differentiation, among several types of sugars. Transcriptome analysis revealed that sucrose suppresses xylem and phloem differentiation in cambial cells. Physiological and genetic analyses suggested that sucrose might function through the BRI1-EMS-SUPPRESSOR1 transcription factor, which is the central regulator of vascular cell differentiation. Conditional overexpression of cytosolic invertase led to a decrease in the number of cambium layers due to an imbalance between cell division and differentiation. Taken together, our results suggest that sucrose potentially acts as a signal that integrates environmental conditions with the developmental program.

Pretreatment with dilute maleic acid enhances the enzymatic digestibility of sugarcane bagasse and oil palm empty fruit bunch fiber.

Lignocellulose is resistant to degradation and requires pretreatment before hydrolytic enzymes can release fermentable sugars. Sulfuric acid has been widely used for biomass pretreatment, but high amount of degradation products usually occurred when using this method. To enhance accessibility to cellulose, we studied the performances of several dilute organic acid pretreatments of sugarcane bagasse and oil palm empty fruit bunch fiber. The results revealed that pretreatment with maleic acid yields the highest xylose and glucose release among other organic acids. The effects of concentration, duration of heating and heating temperature were further studied. Dilute maleic acid 1 % (w/w) pretreatment at 180 °C was the key to its viability as a substitute for sulfuric acid. Moreover, maleic acid did not seem to highly promote the formation of either furfural or 5-HMF in the liquid hydrolysate after pretreatment.

Harnessing originally robust yeast for rapid lactic acid bioproduction without detoxification and neutralization.

Acidic and chemical inhibitor stresses undermine efficient lactic acid bioproduction from lignocellulosic feedstock. Requisite coping treatments, such as detoxification and neutralizing agent supplementation, can be eliminated if a strong microbial host is employed in the process. Here, we exploited an originally robust yeast, Saccharomyces cerevisiae BTCC3, as a production platform for lactic acid. This wild-type strain exhibited a rapid cell growth in the presence of various chemical inhibitors compared to laboratory and industrial strains, namely BY4741 and Ethanol-red. Pathway engineering was performed on the strain by introducing an exogenous LDH gene after disrupting the PDC1 and PDC5 genes. Facilitated by this engineered strain, high cell density cultivation could generate lactic acid with productivity at 4.80 and 3.68 g L-1 h-1 under semi-neutralized and non-neutralized conditions, respectively. Those values were relatively higher compared to other studies. Cultivation using real lignocellulosic hydrolysate was conducted to assess the performance of this engineered strain. Non-neutralized fermentation using non-detoxified hydrolysate from sugarcane bagasse as a medium could produce lactic acid at 1.69 g L-1 h-1, which was competitive to the results from other reports that still included detoxification and neutralization steps in their experiments. This strategy could make the overall lactic acid bioproduction process simpler, greener, and more cost-efficient.

The flocculant Saccharomyces cerevisiae strain gains robustness via alteration of the cell wall hydrophobicity.

When lignocellulosic biomass is utilized as a fermentative substrate to produce biochemicals, the existence of a yeast strain resistant to inhibitory chemical compounds (ICCs) released from the biomass becomes critical. To achieve the purpose, in this study, Saccharomyces yeast strains from a NBRC yeast culture collection were used for exploration and evaluated in two different media containing ICCs that mimic one another but resemble the hydrolysate of real biomass. Among them, S. cerevisiae F118 strain shows robustness upon the fermentation with unique flocculation trait that was strongly responsive to ICC stress. When this strain was cultured in the presence of ICCs, its cell wall hydrophobicity increased dramatically, and reduced significantly when the ICCs were depleted, demonstrating that cell-surface hydrophobicity can also act as an adaptive response to the ICCs. Cells from the strain with the highest cell-wall hydrophobicity displayed progressively stronger flocculation, indicating that the F118 strain is having unique robustness under ICC stress. Gene expression perturbation analysis revealed that mot3 gene encoding regulatory Mot3p from the F118 strain was expressed in response to the concentration of ICCs. This gene was found to control expression of ygp1 gene that encoding Ygp1p, one of cell wall proteins. Deep sequencing analysis revealed that the Mot3p of the F118 strain features a unique insertion and deletion of nucleotides that encode glutamine or asparagine residues, particularly in N-terminal domain, as determined by comparison to the Mot3p sequence from the S288c strain, which was employed as a control strain. Furthermore, the cell wall hydrophobicity of the S288c strain was greatly enhanced and became ICC-responsive after gene swapping with the mot3 gene from the F118 strain. The gene-swapped S288c strain fermented 6-fold faster than the wild-type strain, producing 14.5 g/L of ethanol from 30 g/L of glucose consumed within 24 h in a medium containing the ICCs. These such modifications to Mot3p in unique locations in its sequence have a potential to change the expression of a gene involved in cell wall hydrophobicity and boosted the flocculation response to ICC stress, allowing for the acquisition of extraordinary robustness.

An integrated biorefinery strategy for the utilization of palm-oil wastes.

Each year, the palm oil industry generates a significant amount of biomass residue and effluent waste; both have been identified as significant sources of greenhouse gas (GHG) emissions. This issue poses a severe environmental challenge for the industry due to the possibility of long-term negative effects on human well-being. The palm-oil industry must invest significantly in the technology that is required to resolve these issues and to increase the industry's sustainability. However, current technologies for converting wastes such as lignocellulosic components and effluents into biochemical products are insufficient for optimal utilization. This review discusses the geographical availability of palm-oil biomass, its current utilization routes, and then recommends the development of technology for converting palm-oil biomass into value-added products through an integrated biorefinery strategy. Additionally, this review summarizes the palm oil industry's contribution to achieving sustainable development goals (SDGs) through a circular bioeconomy concept.

Recent advances in lignocellulosic biomass white biotechnology for bioplastics.

Lignocellulosic biomass has great potential as an inedible feedstock for bioplastic synthesis, although its use is still limited compared to current edible feedstocks of glucose and starch. This review focuses on recent advances in the production of biopolymers and biomonomers from lignocellulosic feedstocks with downstream processing and chemical polymer syntheses. In microbial production, four routes composed of existing poly (lactic acid) and polyhydroxyalkanoates (PHAs) and the emerging biomonomers of itaconic acid and aromatic compounds were presented to review present challenges and future perspectives, focusing on the use of lignocellulosic feedstocks. Recently, advances in purification technologies decreased the number of processes and their environmental burden. Additionally, the unique structures and high-performance of emerging lignocellulose-based bioplastics have expanded the possibilities for the use of bioplastics. The sequence of processes provides insight into the emerging technologies that are needed for the practical use of bioplastics made from lignocellulosic biomass.

Bioenergy and Biorefinery: Feedstock, Biotechnological Conversion, and Products.

Biorefinery has been suggested to provide relevant substitutes to a number of fossil products. Feedstocks and conversion technologies have, however, been the bottleneck to the realization of this concept. Herein, feedstocks and bioconversion technologies under biorefinery have been reviewed. Over the last decade, research has shown possibilities of generating tens of new products but only few industrial implementations. This is partly associated with low production yields and poor cost-competitiveness. This review addresses the technical barriers associated with the conversion of emerging feedstocks into chemicals and bioenergy platforms and summarizes the developed biotechnological approaches including advances in metabolic engineering. This summary further suggests possible future advances that would expand the portfolio of biorefinery and speed up the realization of biofuels and biochemicals.

Lipid production by Lipomyces starkeyi using sap squeezed from felled old oil palm trunks.

The ability of oleaginous yeast Lipomyces starkeyi to efficiently produce lipids when cultivated on sap extracted from felled oil palm trunk (OPT) as a novel inexpensive renewable carbon source was evaluated. OPT sap was found to contain approximately 98 g/L glucose and 32 g/L fructose. Batch fermentations were performed using three different OPT sap medium conditions: regular sap, enriched sap, and enriched sap at pH 5.0. Under all sap medium conditions, the cell biomass and lipid production achieved were approximately 30 g/L and 60% (w/w), respectively. L. starkeyi tolerated acidified medium (initial pH ≈ 3) and produced considerable amounts of ethanol as well as xylitol as by-products. The fatty acid profile of L. starkeyi was remarkably similar to that of palm oil, one of the most common vegetable oil feedstock used in biodiesel production with oleic acid as the major fatty acid followed by palmitic, stearic and linoleic acids.

GH-10 and GH-11 Endo-1,4-ß-xylanase enzymes from Kitasatospora sp. produce xylose and xylooligosaccharides from sugarcane bagasse with no xylose inhibition.

A novel strategy for the low-cost, high-yield co-production of xylose and xylooligosaccharides together with no xylose inhibition was developed using a novel heterologous expression of XYN10Ks_480 endo-1,4-ß-xylanase with a ricin-type ß-trefoil type of domain and XYN11Ks_480 endo-1,4-ß-xylanase with a CBM 2 superfamily from the Kitasatospora sp in an actinomycetes expression system. Xylose is the main building block for hemicellulose xylan. Our findings demonstrated high levels of expression and catalytic activity for XYN10Ks_480 during hydrolysis of the extracted xylan of bagasse, and three types of xylan-based substrates were used to produce xylose and xylooligosaccharides. However, hydrolysis by XYN11Ks_480 produced xylooligosaccharides without xylose formation. This study demonstrated how integrating sodium hypochlorite-extracted xylan and enzymatic hydrolysis could provide an alternative strategy for the generation of XOS from lignocellulosic material.

Efficient and Supplementary Enzyme Cocktail from Actinobacteria and Plant Biomass Induction.

Actinobacteria plays a key role in the cycling of organic matter in soils. They secret biomass-degrading enzymes that allow it to produce the unique metabolites that originate in plant biomass. Although past studies have focused on these unique metabolites, a large-scale screening of Actinobacteria is yet to be reported to focus on their biomass-degrading ability. In the present study, a rapid and simple method is constructed for a large-scale screening, and the novel resources that form the plant biomass-degrading enzyme cocktail are identified from 850 isolates of Actinobacteria. As a result, Nonomuraea fastidiosa secretes a biomass degrading enzyme cocktail with the highest enzyme titer, although cellulase activities are lower than a commercially available enzyme. So the rich accessory enzymes are suggested to contribute to the high enzyme titer for a pretreated bagasse with a synergistic effect. Additionally, an optimized cultivation method of biomass induction caused to produce the improved enzyme cocktail indicated strong enzyme titers and a strong synergistic effect. Therefore, the novel enzyme cocktails are selected via the optimized method for large-scale screening, and then the enzyme cocktail can be improved via the optimized production with biomass-induction.

Repeated ethanol fermentation from membrane-concentrated sweet sorghum juice using the flocculating yeast Saccharomyces cerevisiae F118 strain.

The aim of this study was to construct a cost-effective method for repeated bioethanol production using membrane (ultrafiltration permeation and nanofiltration concentration)-concentrated sweet sorghum juice by using flocculent Saccharomyces cerevisiae F118 strain. With low initial dry cell concentrations at around 0.28-0.35 g L-1, the S. cerevisiae F118 strain provided an ethanol titer of 86.19 ±â€¯1.15 g L-1 (theoretical ethanol yield of 70.77%), which was higher than the non-flocculent S. cerevisiae BY4741 strain at 33.92 ±â€¯0.99 g L-1 after 24 h fermentation. This result was correlated with higher gene expressions of the sucrose-hydrolysing enzyme invertase, sugar phosphorylation, and pyruvate-to-ethanol pathways in the F118 strain compared with the BY4741 strain. Sequential fed-batch fermentation was conducted, and the F118 strain was easily separated from the fermentation broth via the formation of flocs and sediment. After the 5th cycle of fermentation with the F118 strain, the ethanol concentration reached 100.37 g L-1.

Xylanase and feruloyl esterase from actinomycetes cultures could enhance sugarcane bagasse hydrolysis in the production of fermentable sugars.

The addition of enzymes that are capable of degrading hemicellulose has a potential to reduce the need for commercial enzymes during biomass hydrolysis in the production of fermentable sugars. In this study, a high xylanase producing actinomycete strain (Kitasatospora sp. ID06-480) and the first ethyl ferulate producing actinomycete strain (Nonomuraea sp. ID06-094) were selected from 797 rare actinomycetes, respectively, which were isolated in Indonesia. The addition (30%, v/v) of a crude enzyme supernatant from the selected strains in sugarcane bagasse hydrolysis with low-level loading (1 FPU/g-biomass) of Cellic® CTec2 enhanced both the released amount of glucose and reducing sugars. When the reaction with Ctec2 was combined with crude enzymes containing either xylanase or feruloyl esterase, high conversion yield of glucose from cellulose at 60.5% could be achieved after 72 h-saccharification.

Effect of inoculum size on single-cell oil production from glucose and xylose using oleaginous yeast Lipomyces starkeyi.

Oleaginous microbes can convert substrates such as carbon dioxide, sugars, and organic acids to single-cell oils (SCOs). Among the oleaginous microorganisms, Lipomyces starkeyi is a particularly well-suited host given its impressive native abilities, including the capability to utilize a wide variety of carbon sources. In this work, the potential of L. starkeyi NBRC10381 to produce SCOs in a synthetically nitrogen-limited mineral medium (-NMM) was investigated by differing the inoculum size using glucose and/or xylose as a carbon source. Fermentation using glucose and xylose as mixed carbon sources generated the highest production of biomass at 40.8 g/L, and achieved a lipid content of 84.9% (w/w). When either glucose or xylose was used separately, the totals for achieved lipid content were 79.6% (w/w) and 85.1% (w/w), respectively. However, biomass production was higher for glucose than for xylose (30.3 vs. 28.7 g/L, respectively). This study describes the first simultaneous achievement of higher levels of cell mass and lipid production using glucose and/or xylose as the carbon sources in different inoculum sizes.

Glutathione production from mannan-based bioresource by mannanase/mannosidase expressing Saccharomyces cerevisiae.

This work aims to produce glutathione directly from mannan-based bioresources using engineered Saccharomyces cerevisiae. Mannan proved to be a valuable carbon source for glutathione production by this organism. Mannan-hydrolyzing S. cerevisiae was developed by heterologous expression of mannanase/mannosidase on its cell surface. This strain efficiently produced glutathione from mannose polysaccharide, ß-1,4-mannan. Furthermore, it produced glutathione from locust bean gum (LBG), a highly dense and inexpensive mannan-based bioresource, as sole carbon source. Glutathione productivity from LBG was enhanced by engineering the glutathione metabolism of mannan-hydrolyzing S. cerevisiae. Expression of extracellular mannanase/mannosidase protein combined with intracellular metabolic engineering is potentially applicable to the efficient, environmentally friendly bioproduction of targeted products from mannan-based bioresources.

Challenges of non-flocculating Saccharomyces cerevisiae haploid strain against inhibitory chemical complex for ethanol production.

This study provides insight observation based on the gene expression and the metabolomic analysis of the natural robust yeast Saccharomyces cerevisiae NBRC849 during the fermentation in the medium containing inhibitory chemical complexes (ICC) at different concentrations. The tolerance mechanisms involved in the strain might have existed through the upregulation of genes involved in NAD(H)/NADP(H) cofactors generations (ALD6, ZWF1, GND1), membrane robustness for efflux pump (YOR1, PDR5, TPO3) and cation/polyamine transport (TPO3). The alteration of metabolic flux to the shikimic pathway was also found in this strain, resulted in the enhanced formation of aromatic amino acid required for cell survival. Enhanced expression of these genes as well as the increase of metabolic flux to shikimic pathway were suggested to result in the robustness of non-flocculating S. cerevisiae haploid strain.

Mannan endo-1,4-ß-mannosidase from Kitasatospora sp. isolated in Indonesia and its potential for production of mannooligosaccharides from mannan polymers.

Mannan endo-1,4-ß-mannosidase (commonly known as ß-mannanase) catalyzes a random cleavage of the ß-D-1,4-mannopyranosyl linkage in mannan polymers. The enzyme has been utilized in biofuel production from lignocellulose biomass, as well as in production of mannooligosaccharides (MOS) for applications in feed and food industries. We aimed to obtain a ß-mannanase, for such mannan polymer utilization, from actinomycetes strains isolated in Indonesia. Strains exhibiting high mannanase activity were screened, and one strain belonging to the genus Kitasatospora was selected. We obtained a ß-mannanase from this strain, and an amino acid sequence of this Kitasatospora ß-mannanase showed a 58-71% similarity with the amino acid sequences of Streptomyces ß-mannanases. The Kitasatospora ß-mannanase showed a significant level of activity (944 U/mg) against locust bean gum (0.5% w/v) and a potential for oligosaccharide production from various mannan polymers. The ß-mannanase might be beneficial particularly in the enzymatic production of MOS for applications of mannan utilization.