Adaptive laboratory evolution and reverse engineering enhances autotrophic growth in Pichia pastoris

Synthetic biology offers several routes for CO₂ conversion into biomass or bio-chemicals, helping to avoid unsustainable use of organic feedstocks, which negatively contribute to climate change. The use of well-known industrial organisms, such as the methylotrophic yeast Pichia pastoris (Komagataella phaffii), for the establishment of novel C1-based bioproduction platforms could wean biotechnology from feedstocks with alternative use in food production. Recently, the central carbon metabolism of P. pastoris was re-wired following a rational engineering approach, allowing the resulting strains to grow autotrophically with a μ_max of 0.008 h⁻¹, which was further improved to 0.018 h⁻¹ by adaptive laboratory evolution. Using reverse genetic engineering of single-nucleotide (SNPs) polymorphisms occurring in the genes encoding for phosphoribulokinase and nicotinic acid mononucleotide adenylyltransferase after evolution, we verified their influence on the improved autotrophic phenotypes. The reverse engineered SNPs lead to lower enzyme activities in putative branching point reactions and in reactions involved in energy balancing. Beyond this, we show how further evolution facilitates peroxisomal import and increases growth under autotrophic conditions. The engineered P. pastoris strains are a basis for the development of a platform technology, which uses CO₂ for production of value-added products, such as cellular biomass, technical enzymes and chemicals and which further avoids consumption of organic feedstocks with alternative use in food production. Further, the identification and verification of three pivotal steps may facilitate the integration of heterologous CBB cycles or similar pathways into heterotrophic organisms.     

The real objective of Mendel's paper: A response to Monaghan and Corcos

Mendel's work in hybridization is ipso facto a study in inheritance. He is explicit in his interest to formulate universal generalizations, and at least in the case of the independent segregation of traits, he formulated his conclusions in the form of a law. Mendel did not discern, however, the inheritance of traits from that of the potential for traits. Choosing to study discrete non-overlapping traits, this did not hamper his efforts.     

Isolation and characterization of a methyl chloride utilizing,strictly anaerobic bacterium

From sludge obtained from the sewage digester plant in Stuttgart-Möhringen a strictly anaerobic bacterium was enriched and isolated with methyl chloride as the energy source. The isolate, which was tentatively called strain MC, was nonmotile, gram-positive, and occurred as elongated cocci arranged in chains. Cells of strain MC formed about 3 mol of acetate per 4 mol of CH₃Cl consumed, indicating that the organism was a homoacetogenic bacterium fermenting methyl chloride plus CO₂ according to: The organism grew with 2–3% methyl chloride in the gas phase at a doubling time of near 30 h. Dichloromethane was not utilized. The bacterium also grew on carbon monoxide, H₂ plus CO₂, and methoxylated aromatic compounds. Optimal growth with methyl chloride was observed at 25°C and pH 7.3–7.7. The G+C-content of the DNA was 47.5±1.5%. The methyl chloride conversion appeared to be inducible, since H₂ plus CO₂-grown cells lacked this ability. From the morphological and physiological characteristics, the isolate could not be affiliated to a known species.     

Effects of potassium levels over the range occurring in host plants on the growth of an insect herbivore (Manduca sexta)

Third instar tobacco hornworms (Manduca sexta L.: Sphingidae) on low dietary potassium had a lower relative growth rate than individuals on diets with potassium concentrations reflecting those in host-plants, due to decreased consumption rate, lower efficiencies of conversion of ingested and digested food (ECI and ECD), and a prolonged growth/feeding phase. Furthermore, these larvae, when placed on a diet with a moderate potassium concentration through the fourth stadium, ended up being smaller due to lower ECI and less biomass gained, and had a prolonged growth phase, which suggest an irreversible cost of the previous low potassium diet. Third instar hornworms on high potassium diets had lower ECI and ECD, and they had a prolonged growth phase. These individuals, when placed on a moderate potassium diet in the fourth stadium, gained less biomass, than those previously offered hostplant-like-potassium diets. Body potassium concentrations (% dw) at the end of the third stadium were similar among treatment groups. With increasing potassium concentrations in the diet, utilization efficiencies of potassium decreased and potassium concentrations in the frass increased. Correspondingly, water content (% fw) of the newly-molted fourth instar larvae declined with increasing potassium, indicating a passive loss of water during potassium excretion. Low and high dietary potassium reduced survivorship of third instar larvae; fourth instar caterpillars previously fed the low potassium diet also had poor survivorship. We conclude that, within the normal range of potassium concentrations in the hostplants, caterpillar performance is largely unaffected by potassium concentration, but that potassium-poor and potassium-rich diets, such as those hornworms may sometimes experience, can reduce growth and survivorship.     

Associative N2-fixation in plants growing in saline sodic soils and its relative quantification based on15N natural abundance

The interactive effect of low P supply (0, 10, 20 and 40 M) and plant age on nodule number, mass and functioning (ureide analysis technique), vegetative growth and pod production were investigated in glasshouse-grown nodulated cowpea (Vigna unguiculata L.cv. Kausband) in sand culture. Compared with 40 M P, P stress (0 M P) or very low (10 M P) supply markedly impaired nodulation, allantoin and amino-N concentrations and weight of N solutes in xylem exudates. Consequently, P stress reduced top growth and pod yields by 48 and 90%, respectively. N solutes in xylem exudates and total plant N assayed by Kjeldahl technique (as estimates of N₂ fixation) responded similarly to P supply. However, the relative ureide index [(ureide-N/ureide N+amino-N)×100] remained constant (99%), irrespective of P supply, indicating the plants' complete dependency on symbiosis for growth, without implying that growth was markedly increased by N₂ fixation. Although P concentrations in plant tops, roots and nodules increased with P supply, N concentrations in these plant tissues were unaffected by P supply. The concentrations of N and P in the nodules were 2–2 1/2 times higher than in plant tops. P application interacted strongly with plant age, with the largest P effect evidently achieved at the early podding stage. The significance and implications of these results are discussed.     

气候变化对内蒙古中部草原优势牧草生长季的影响

研究温性典型草原优势牧草生长季与气候因子变化的关系,对于监测草地环境变化及保护利用,评估区域气候变化对优势牧草生长的影响,指导畜牧业生产等具有重要的科学意义和实践价值.基于内蒙古中部草原3个牧业气象试验站1983-2009年克氏针茅和羊草的物候期、气象资料,对返青、黄枯日期、生长季与气温、降水量之间的关系进行了统计分析.结果表明:(1)27a间,该区的年、春季、夏季和秋季平均气温均呈显著升高趋势,春季升温幅度最大,气候倾向率为0.71-0.84℃/10a;各时段降水量变化除春季呈微量增加趋势外,总体呈减少态势.可见,内蒙古中部温性典型草原区暖干化趋势明显.(2)27a间,锡林浩特克氏针茅和羊草返青日期表现为推后趋势,并与4月降水量呈显著正相关;镶黄旗和察右后旗克氏针茅和羊草返青日期呈提前趋势,并与3-5月气温、降水量呈显著负相关,气温每升高1℃、降水量每增加10mm,优势牧草返青日期约提前3.0-5.1d和1.3-2.1d.(3)该区克氏针茅和羊草黄枯日期均呈提前趋势,与8-9月气温均呈显著负相关,降水量为正相关,气温每升高1℃,克氏针茅黄枯日期提前1.7-10.7 d,羊草黄枯日期提前3.5-11.3d,降水量的影响相对较弱.(4)该区克氏针茅生长季延长趋势明显,羊草生长季缩短趋势明显.克氏针茅生长季与4-10月均温正相关系数最大,羊草生长季与水热系数的正相关系数最大.     

生物催化法合成6-氰基-（3R，5R）-二羟基己酸叔丁酯

利用E.coli BL21／pCDFDuet-gdh—cr-X共表达全细胞催化6-氰基-（5R）-羟基-3-羰基己酸叔丁酯不对称还原合成6-氰基-（3R,5R）-二羟基已酸叔丁酯。结果表明：在菌体用量4．85g/L、葡萄糖与底物质量浓度比为1：1、温度28℃、pH7．0条件下,80．0g／L6-氰基-（5R）-羟基-3-羰基己酸叔丁酯生物还原2h后,底物转化率可达99．0％,产物d.e．值大于99．5％。在考察范围内,NADP^＋用量对催化效率无显著作用。     

Differences in the Optical Response of MSU and CPP Crystals During Magnetic Orientation: Possibility of Diagnosing Gout and Pseudogout

Pseudogout is crystalline arthritis. It has a similar clinical picture to that of gout, and it is difficult to distinguish the two diseases using conventional analysis methods. However, it is important to identify the different crystals responsible for these two cases because the treatment strategies are different. In a previous study, we reported magnetic orientation of monosodium urate (MSU) crystals, which are the causative agent of gout, at the permanent magnet level. In this study, we investigated the effect of an applied magnetic field on calcium pyrophosphate (CPP) crystals, which are the causative agent of pseudogout, and the difference in the magnetic responses of CPP and MSU crystals. We found that the CPP crystals were oriented in a magnetic field on milli-Tesla order because of the anisotropy of the diamagnetic susceptibility. In addition, the CPP crystals exhibited different anisotropic magnetic properties from those of MSU crystals, which led to a characteristic difference between the orientations of the two crystals. That is, we found that the causative agents of gout and pseudogout responded differently to a magnetic field. This report suggests that the discrimination between CPP and MSU by optical measurements is possible by application of magnetic fields appropriately. © 2023 Bioelectromagnetics Society.