Enhancing L-malate production of Aspergillus oryzae by nitrogen regulation strategy.

Regulating morphology engineering and fermentation of Aspergillus oryzae makes it possible to increase the titer of L-malate. However, the existing L-malate-producing strain has limited L-malate production capacity and the fermentation process is insufficiently mature, which cannot meet the needs of industrial L-malate production. To further increase the L-malate production capacity of A. oryzae, we screened out a mutant strain (FMME-S-38) that produced 79.8 g/L L-malate in 250-mL shake flasks, using a newly developed screening system based on colony morphology on the plate. We further compared the extracellular nitrogen (N1) and intracellular nitrogen (N2) contents of the control and mutant strain (FMME-S-38) to determine the relationship between the curve of nitrogen content (N1 and N2) and the L-malate titer. This correlation was then used to optimize the conditions for developing a novel nitrogen supply strategy (initial tryptone concentration of 6.5 g/L and feeding with 3 g/L tryptone at 24 h). Fermentation in a 7.5-L fermentor under the optimized conditions further increased the titer and productivity of L-malate to 143.3 g/L and 1.19 g/L/h, respectively, corresponding to 164.9 g/L and 1.14 g/L/h in a 30-L fermentor. This nitrogen regulation-based strategy cannot only enhance industrial-scale L-malate production but also has generalizability and the potential to increase the production of similar metabolites.Key Points• Construction of a new screening system based on colony morphology on the plate.• A novel nitrogen regulation strategy used to regulate the production of L-malate.• A nitrogen supply strategy used to maximize the production of L-malate.

Consistent behavioral and electrophysiological evidence for rapid perceptual discrimination among the six human basic facial expressions.

The extent to which the six basic human facial expressions perceptually differ from one another remains controversial. For instance, despite the importance of rapidly decoding fearful faces, this expression often is confused with other expressions, such as Surprise in explicit behavioral categorization tasks. We quantified implicit visual discrimination among rapidly presented facial expressions with an oddball periodic visual stimulation approach combined with electroencephalography (EEG), testing for the relationship with behavioral explicit measures of facial emotion discrimination. We report robust facial expression discrimination responses bilaterally over the occipito-temporal cortex for each pairwise expression change. While fearful faces presented as repeated stimuli led to the smallest deviant responses from all other basic expressions, deviant fearful faces were well discriminated overall and to a larger extent than expressions of Sadness and Anger. Expressions of Happiness did not differ quantitatively as much in EEG as for behavioral subjective judgments, suggesting that the clear dissociation between happy and other expressions, typically observed in behavioral studies, reflects higher-order processes. However, this expression differed from all others in terms of scalp topography, pointing to a qualitative rather than quantitative difference. Despite this difference, overall, we report for the first time a tight relationship of the similarity matrices across facial expressions obtained for implicit EEG responses and behavioral explicit measures collected under the same temporal constraints, paving the way for new approaches of understanding facial expression discrimination in developmental, intercultural, and clinical populations.

Enhancement of Sphingolipid Synthesis Improves Osmotic Tolerance of Saccharomyces cerevisiae.

To enhance the growth performance of Saccharomyces cerevisiae under osmotic stress, mutant XCG001, which tolerates up to 1.5 M NaCl, was isolated through adaptive laboratory evolution (ALE). Comparisons of the transcriptome data of mutant XCG001 and the wild-type strain identified ELO2 as being associated with osmotic tolerance. In the ELO2 overexpression strain (XCG010), the contents of inositol phosphorylceramide (IPC; t18:0/26:0), mannosylinositol phosphorylceramide [MIPC; t18:0/22:0(2OH)], MIPC (d18:0/22:0), MIPC (d20:0/24:0), mannosyldiinositol phosphorylceramide [M(IP)2C; d20:0/26:0], M(IP)2C [t18:0/26:0(2OH)], and M(IP)2C [d20:0/26:0(2OH)] increased by 88.3 times, 167 times, 63.3 times, 23.9 times, 27.9 times, 114 times, and 208 times at 1.0 M NaCl, respectively, compared with the corresponding values of the control strain XCG002. As a result, the membrane integrity, cell growth, and cell survival rate of strain XCG010 increased by 24.4% ± 1.0%, 21.9% ± 1.5%, and 22.1% ± 1.1% at 1.0 M NaCl, respectively, compared with the corresponding values of the control strain XCG002 (wild-type strain with a control plasmid). These findings provided a novel strategy for engineering complex sphingolipids to enhance osmotic tolerance.IMPORTANCE This study demonstrated a novel strategy for the manipulation of membrane complex sphingolipids to enhance S. cerevisiae tolerance to osmotic stress. Elo2, a sphingolipid acyl chain elongase, was related to osmotic tolerance through transcriptome analysis of the wild-type strain and an osmosis-tolerant strain generated from ALE. Overexpression of ELO2 increased the content of complex sphingolipid with longer acyl chain; thus, membrane integrity and osmotic tolerance improved.

Pathway engineering of Escherichia coli for α-ketoglutaric acid production.

α-Ketoglutaric acid (α-KG) is a multifunctional dicarboxylic acid in the tricarboxylic acid (TCA) cycle, but microbial engineering for α-KG production is not economically efficient, due to the intrinsic inefficiency of its biosynthetic pathway. In this study, pathway engineering was used to improve pathway efficiency for α-KG production in Escherichia coli. First, the TCA cycle was rewired for α-KG production starting from pyruvate, and the engineered strain E. coli W3110Δ4-PCAI produced 15.66 g/L α-KG. Then, the rewired TCA cycle was optimized by designing various strengths of pyruvate carboxylase and isocitrate dehydrogenase expression cassettes, resulting in a large increase in α-KG production (24.66 g/L). Furthermore, acetyl coenzyme A (acetyl-CoA) availability was improved by overexpressing acetyl-CoA synthetase, leading to α-KG production up to 28.54 g/L. Finally, the engineered strain E. coli W3110Δ4-P(H) CAI(H) A was able to produce 32.20 g/L α-KG in a 5-L fed-batch bioreactor. This strategy described here paves the way to the development of an efficient pathway for microbial production of α-KG.

Engineering of membrane phospholipid component enhances salt stress tolerance in Saccharomyces cerevisiae.

To increase the growth of industrial strains under environmental stress, the Saccharomyces cerevisiae BY4741 salt-tolerant strain Y00 that tolerates 1.2 M NaCl was cultured through nitroguanidine mutagenesis. The metabolomics and transcription data of Y00 were compared with those of the wild-type strain BY4741. The comparison identified two genes related to salt stress tolerance, cds1 and cho1. Modular assembly of cds1 and cho1 redistributed the membrane phospholipid component and decreased the ratio of anionic-to-zwitterionic phospholipid in strain Y03 that showed the highest salt tolerance. Therefore, significantly increased membrane potential and membrane integrity helped strain Y03 to resist salt stress (1.2 M NaCl). This study provides an effective membrane engineering strategy to enhance salt stress tolerance.

Improving lysine production through construction of an Escherichia coli enzyme-constrained model.

Microbial cell factories are widely used for the production of high-value chemicals. However, maximizing production titers is made difficult by the complicated regulatory mechanisms of these cell platforms. Here, kcat values were incorporated to construct an Escherichia coli enzyme-constrained model. The resulting ec_iML1515 model showed that the protein demand and protein synthesis rate were the key factors affecting lysine production. By optimizing the expression of the 20 top-demanded proteins, lysine titers reached 95.7 ± 0.7 g/L, with a 0.45 g/g glucose yield. Moreover, adjusting NH4+ and dissolved oxygen levels to regulate the synthesis rate of energy metabolism-related proteins caused lysine titers and glucose yields to increase to 193.6 ± 1.8 g/L and 0.74 g/g, respectively. The ec_iML1515 model provides insight into how enzymes required for the biosynthesis of certain products are distributed between and within metabolic pathways. This information can be used to accurately predict and rationally design lysine production.

Engineering protonation conformation of l-aspartate-α-decarboxylase to relieve mechanism-based inactivation.

Mechanism-based inactivation of l-aspartate-α-decarboxylase (PanD), which leads to irreversible modification of active site, is a major challenge in the efficient production of ß-alanine from L-aspartic acid. In this study, a semi-rational strategy that combined conformational dynamics and structural alignment was applied to increase the catalytic stability of Bacillus subtilis PanD (BsPanD). Using site-saturation and C-terminal deletion, the variant Q5 (BsPanDI46V/I88M/K104S/I126* ) was generated. The catalytic half-life and the total turnover number (TTN) of Q5 were 3.48-fold and 2.52-fold higher, respectively, compared with that of the parent Q0. The reasons for the differences were the prolonged distance d1 between the phenolic group of Tyr58 and pyruvoyl group of Ser25 (4.9 Å in Q0 vs. 5.5 Å in Q5), an increased difficulty for incorrect protonation to occur, and the decreased flexibility of residues in regions A, B, and C, thereby enhancing the probability of correct protonation. Variant Q5, coupled with l-aspartase (AspA) in a 15-L bioreactor, generated a linear cascade system using fumaric acid as a substrate, yielding 118.6 g/L ß-alanine with a product/catalyst (P/C) ratio of 5.9 g/g and a conversion > 99%. These results showed that reshaping the protonation conformation of PanD can efficiently relieve mechanism-based inactivation and boost catalytic stability.

DCEO Biotechnology: Tools To Design, Construct, Evaluate, and Optimize the Metabolic Pathway for Biosynthesis of Chemicals.

Chemical synthesis is a well established route for producing many chemicals on a large scale, but some drawbacks still exist in this process, such as unstable intermediates, multistep reactions, complex process control, etc. Biobased production provides an attractive alternative to these challenges, but how to make cells into efficient factories is challenging. As a key enabling technology to develop efficient cell factories, design-construction-evaluation-optimization (DCEO) biotechnology, which incorporates the concepts and techniques of pathway design, pathway construction, pathway evaluation, and pathway optimization at the systems level, offers a conceptual and technological framework to exploit potential pathways, modify existing pathways and create new pathways for the optimal production of desired chemicals. Here, we summarize recent progress of DCEO biotechnology and examples of its application, and provide insights as to when, what and how different strategies should be taken. In addition, we highlight future perspectives of DCEO biotechnology for the successful establishment of biorefineries.

Morphology engineering of Aspergillus oryzae for l-malate production.

Aspergillus oryzae is a competitive natural producer for organic acids, but its production capacity is closely correlated with a specific morphological type. Here, morphology engineering was used for tailoring A. oryzae morphology to enhance l-malate production. Specifically, correlation between A. oryzae morphology and l-malate fermentation was first conducted, and the optimal range of the total volume of pellets in a unit volume of fermentation broth (V value) for l-malate production was 120-130 mm3 /ml. To achieve this range, A. oryzae morphology was improved by controlling the variation of operational parameters, such as agitation speed and aeration rate, and engineered by optimizing the expression of cell division cycle proteins such as tyrosine-protein phosphatase (CDC14), anaphase promoting complex/cyclosome activator protein (CDC20), and cell division control protein 45 (CDC45). By controlling the strength of CDC14 at a medium level, V value fell into the optimal range of V value and the final engineered strain A. oryzae CDC14(3) produced up to 142.5 g/L l-malate in a 30-L fermenter. This strategy described here lays a good foundation for industrial production of l-malate in the future, and opens a window to develop filamentous fungi as cell factories for production of other chemicals.

Enhancing L-malate production of Aspergillus oryzae FMME218-37 by improving inorganic nitrogen utilization.

Microbial L-malate production from renewable feedstock is a promising alternative to petroleum-based chemical synthesis. However, high L-malate production of Aspergillus oryzae was achieved to date using organic nitrogen, with inorganic nitrogen still unable to meet industrial applications. In the current study, we constructed a screening system and nitrogen supply strategy to improve L-malate production with ammonium sulphate [(NH4)2SO4] as the sole nitrogen source. First, we generated and identified a high-producing mutant FMME218-37, which stably boosted L-malate production from 30.73 to 78.12 g/L, using a combined screening system with morphological characteristics. Then, by analyzing the fermentation parameters and physiological characteristics, we further speculated the key factor was the unbalance of carbon and nitrogen absorption. Finally, the titer and productivity of L-malate was increased to 95.2 g/L and 0.57 g/(L h) by regulating the nitrogen supply module to balance carbon and nitrogen absorption, which represented the highest level in A. oryzae with (NH4)2SO4 as nitrogen source achieved to date. Moreover, our findings using a low-cost substrate may lead to building an economical cell factory of A. oryzae for L-malate production.

Promoter engineering of cascade biocatalysis for α-ketoglutaric acid production by coexpressing L-glutamate oxidase and catalase.

Enzymatic transformation is now an attractive alternative for α-ketoglutaric acid (α-KG) production, but the oxidative deamination from L-glutamic acid to α-KG is along with H2O2 accumulation. To remove the effect of H2O2 on α-KG production, in vitro cascade biocatalysis was designed using the purified L-glutamate oxidase (LGOX) from Streptomyces ghanaensis and catalase (KatG) from Escherichia coli W3110, and the optimal ratio of LGOX:KatG (2.0:1250) was detected in this system. To achieve this ratio, in vivo cascade biocatalysis was constructed by varying promoters and ribosome binding sites (RBSs), and optimized by promoter engineering, such as adjusting the junctions between the SD sequence and start codon ATG and tuning the strengths of RBSs. When the final ratio of LGOX:KatG (2.1:1185) in strain E. coli-(T7)LGOX-(rbs2)KatG was used, α-KG concentration and its conversion rate were increased to 106 g L-1 and 96%, respectively. This strategy described here paves the way to the development of cascade biocatalysis for enzymatic production of other chemicals.

Enhanced pyruvate production in Candida glabrata by overexpressing the CgAMD1 gene to improve acid tolerance.

OBJECTIVES: To enhance acid tolerance of Candida glabrata for pyruvate production by engineering AMP metabolism. RESULTS: The physiological function of AMP deaminase in AMP metabolism from C. glabrata was investigated by deleting or overexpresseing the corresponding gene, CgAMD1. At pH 4, CgAMD1 overexpression resulted in 59 and 51% increases in biomass and cell viability compared to those of wild type strain, respectively. In addition, the intracellular ATP level of strain Cgamd1Δ/CgAMD1 was down-regulated by 22%, which led to a 94% increase in pyruvate production. Further, various strengths of CgAMD1 expression cassettes were designed, thus resulting in a 59% increase in pyruvate production at pH 4. Strain Cgamd1Δ/CgAMD1 (H) was grown in a 30 l batch bioreactor at pH 4, and pyruvate reached 46.1 g/l. CONCLUSION: CgAMD1 overexpression plays an active role in improving acid tolerance and pyruvate fermentation performance of C. glabrata at pH 4.

[Direct determination of isocarbophos by using oil-soluble CdSe quantum dots as fluorescence probe].

A simple, rapid and direct approach to isocarbophos determination was developed based on the fluorescence quenching of oil-soluble CdSe quantum dots by isocarbophos. Under the optimized conditions, the linear range of oil-soluble CdSe QDs fluorescence intensity versus the concentration of isocarbophos was 2.30 x 10(-1) - 1.09 x 10(-5) mol x L(-1), with a correlation coefficient of 0.999 9 and a limit of detection of 1.1 x 10(-7) mol x L(-1). The proposed method has been applied to the determination of isocarbophos in rice and wheat flour with standard recoveries of 93.3%-105.0%. The fluorescence quenching mechanism was further investigated by using UV-Vis absorption spectra and time-resolved fluorescence spectrum. The results indicated that the fluorescence quenching of oil-soluble CdSe QDs by isocarbophos was caused by the change of the surface state which resulted in the enhancement of the surface defect and the occurrence of the non-radiational recombination.

CRK41 Modulates Microtubule Depolymerization in Response to Salt Stress in Arabidopsis.

The pivotal role of cysteine-rich receptor-like kinases (CRKs) in modulating growth, development, and responses to stress has been widely acknowledged in Arabidopsis. However, the function and regulation of CRK41 has remained unclear. In this study, we demonstrate that CRK41 is critical for modulating microtubule depolymerization in response to salt stress. The crk41 mutant exhibited increased tolerance, while overexpression of CRK41 led to hypersensitivity to salt. Further analysis revealed that CRK41 interacts directly with the MAP kinase3 (MPK3), but not with MPK6. Inactivation of either MPK3 or MPK6 could abrogate the salt tolerance of the crk41 mutant. Upon NaCl treatment, microtubule depolymerization was heightened in the crk41 mutant, yet alleviated in the crk41mpk3 and crk41mpk6 double mutants, indicating that CRK41 suppresses MAPK-mediated microtubule depolymerizations. Collectively, these results reveal that CRK41 plays a crucial role in regulating microtubule depolymerization triggered by salt stress through coordination with MPK3/MPK6 signalling pathways, which are key factors in maintaining microtubule stability and conferring salt stress resistance in plants.

The Neural Signature of Social Dominance Discrimination by Means of Fast Periodic Visual Stimulation.

Hierarchy is a pervasive feature of social organization. The ability to rapidly discriminate hierarchical information is critical for social interaction. Here, we took advantage of a special technique in electroencephalography (EEG) known as fast periodic visual stimulation (FPVS). We used this technique, which captures the automatic perception of faces, to explore the neural signature of social dominance discrimination. A stream of computer-generated faces was presented at 6 Hz, i.e. six faces/second. In the experimental condition, faces alternated from high to low social dominance within a sequence, bringing about a frequency of interest of 3 Hz (6 Hz/2), i.e. three high/low dominance faces appeared in one second. In two control conditions, we presented faces which came exclusively from one of two hierarchical ranks (either lower or higher). Participants were asked to respond to information unrelated to this hierarchical information, namely pressing the spacebar when the fixation changes color. Results revealed a significant 3 Hz response for the experimental condition only. This response was located bilaterally in the occipito-temporal region, indicating discrimination of differences in social dominance. Through the use of FPVS, we provide electrophysiological evidence to show that social hierarchical information can be detected automatically.

Enzymatic production of trans-4-hydroxy-l-proline by proline 4-hydroxylase.

Trans-4-hydroxy-l-proline (Hyp) is a useful chiral building block for production of many nutritional supplements and pharmaceuticals. However, it is still challenging for industrial production of Hyp due to heavy environmental pollution and low production efficiency. To establish a green and efficient process for Hyp production, the proline 4-hydroxylase (DsP4H) from Dactylosporangium sp. RH1 was overexpressed and functionally characterized in Escherichia coli BL21(DE3). The recombinant DsP4H with l-proline as a substrate exhibited Km , kcat and kcat /Km values up to 0.80 mM, 0.52 s-1 and 0.65 s-1 ·mM-1 respectively. Furthermore, DsP4H showed the highest activity at 35°C and pH 6.5 towards l-proline. The highest enzyme activity of 175.6 U mg-1 was achieved by optimizing culture parameters. Under the optimal transformation conditions in a 5-l fermenter, Hyp titre, conversion rate and productivity were up to 99.9 g l-1 , 99.9% and 2.77 g l-1  h-1 respectively. This strategy described here provides an efficient method for production of Hyp and thus has a great potential in industrial application.

Chassis engineering of Escherichia coli for trans-4-hydroxy-l-proline production.

Microbial production of trans-4-hydroxy-l-proline (Hyp) offers significant advantages over conventional chemical extraction. However, it is still challenging for industrial production of Hyp due to its low production efficiency. Here, chassis engineering was used for tailoring Escherichia coli cellular metabolism to enhance enzymatic production of Hyp. Specifically, four proline 4-hydroxylases (P4H) were selected to convert l-proline to Hyp, and the recombinant strain overexpressing DsP4H produced 32.5 g l-1 Hyp with α-ketoglutarate addition. To produce Hyp without α-ketoglutarate addition, α-ketoglutarate supply was enhanced by rewiring the TCA cycle and l-proline degradation pathway, and oxygen transfer was improved by fine-tuning heterologous haemoglobin expression. In a 5-l fermenter, the engineered strain E. coliΔsucCDΔputA-VHb(L) -DsP4H showed a significant increase in Hyp titre, conversion rate and productivity up to 49.8 g l-1 , 87.4% and 1.38 g l-1  h-1 respectively. This strategy described here provides an efficient method for production of Hyp, and it has a great potential in industrial application.

[Application of chronological lifespan in the construction of Escherichia coli cell factories].

Microbial cell factories capable of producing valuable chemicals from renewable feedstocks provide a promising alternative towards sustainability. However, environmental stress remarkably affects the performance of microbial cell factories. By extending the chronological lifespan of microbial cells, the performance of microbial cell factories can be greatly improved. Firstly, an evaluation system for chronological lifespan and semi-chronological lifespan was established based on the changes in survival rates. Secondly, the addition of anti-aging drugs such as cysteine, carnosine, aminoguanidine and glucosamine increased the chronological lifespan of E. coli by 80%, 80%, 50% and 120%, respectively. Finally, we demonstrated that extending the chronological lifespan of E. coli increased the yield of metabolites produced by E. coli cell factories with endogenous (lactic acid and pyruvic acid) or exogenous (malic acid) metabolic pathway by 30.0%, 25.0%, and 27.0%, respectively. The strategy of extending chronological lifespan of E. coli provides a potential approach for enhancing the performance of microbial cell factories.

Engineering the transmission efficiency of the noncyclic glyoxylate pathway for fumarate production in Escherichia coli.

BACKGROUND: Fumarate is a multifunctional dicarboxylic acid in the tricarboxylic acid cycle, but microbial engineering for fumarate production is limited by the transmission efficiency of its biosynthetic pathway. RESULTS: Here, pathway engineering was used to construct the noncyclic glyoxylate pathway for fumarate production. To improve the transmission efficiency of intermediate metabolites, pathway optimization was conducted by fluctuating gene expression levels to identify potential bottlenecks and then remove them, resulting in a large increase in fumarate production from 8.7 to 16.2 g/L. To further enhance its transmission efficiency of targeted metabolites, transporter engineering was used by screening the C4-dicarboxylate transporters and then strengthening the capacity of fumarate export, leading to fumarate production up to 18.9 g/L. Finally, the engineered strain E. coli W3110△4-P(H)CAI(H)SC produced 22.4 g/L fumarate in a 5-L fed-batch bioreactor. CONCLUSIONS: In this study, we offered rational metabolic engineering and flux optimization strategies for efficient production of fumarate. These strategies have great potential in developing efficient microbial cell factories for production of high-value added chemicals.

A robust implicit measure of facial attractiveness discrimination.

Decisions of attractiveness from the human face are made instantly and spontaneously, but robust implicit neural measures of facial attractiveness discrimination are currently lacking. Here we applied fast periodic visual stimulation coupled with electroencephalography (EEG) to objectively measure the neural coding of facial attractiveness. We presented different pictures of faces at 6 Hz, i.e. six faces/second, for a minute while participants attended to a central fixation cross and indicated whether the cross shortly changed color. Every other face in the stimulation was attractive and was replaced by a relatively less attractive face. This resulted in alternating more/less attractive faces at a 3 Hz rate, eliciting a significant increase in occipito-temporal EEG amplitude at 3 Hz both at the group and the individual participant level. This response was absent in two control conditions where either only attractive or only less attractive faces were presented. These observations support the view that face-sensitive visual areas discriminate attractiveness implicitly and rapidly from the human face.