A Library of Aspergillus niger Chassis Strains for Morphology Engineering Connects Strain Fitness and Filamentous Growth With Submerged Macromorphology

Submerged fermentation using filamentous fungal cell factories is used to produce a diverse portfolio of useful molecules, including food, medicines, enzymes, and platform chemicals. Depending on strain background and abiotic culture conditions, different macromorphologies are formed during fermentation, ranging from dispersed hyphal fragments to approximately spherical pellets several millimetres in diameter. These macromorphologies are known to have a critical impact on product titres and rheological performance of the bioreactor. Pilot productivity screens in different macromorphological contexts is technically challenging, time consuming, and thus a significant limitation to achieving maximum product titres. To address this bottleneck, we developed a library of conditional expression mutants in the organic, protein, and secondary metabolite cell factory Aspergillus niger. Thirteen morphology-associated genes transcribed during fermentation were placed via CRISPR-Cas9 under control of a synthetic Tet-on gene switch. Quantitative analysis of submerged growth reveals that these strains have distinct and titratable macromorphologies for use as chassis during strain engineering programs. We also used this library as a tool to quantify how pellet formation is connected with strain fitness and filamentous growth. Using multiple linear regression modelling, we predict that pellet formation is dependent largely on strain fitness, whereas pellet Euclidian parameters depend on fitness and hyphal branching. Finally, we have shown that conditional expression of the putative kinase encoding gene pkh2 can decouple fitness, dry weight, pellet macromorphology, and culture heterogeneity. We hypothesize that further analysis of this gene product and the cell wall integrity pathway in which it is embedded will enable more precise engineering of A. niger macromorphology in future.

Changes in Oxygen Availability during Glucose-Limited Chemostat Cultivations of Penicillium chrysogenum Lead to Rapid Metabolite, Flux and Productivity Responses

Bioreactor scale-up from the laboratory scale to the industrial scale has always been a pivotal step in bioprocess development. However, the transition of a bioeconomy from innovation to commercialization is often hampered by performance loss in titer, rate and yield. These are often ascribed to temporal variations of substrate and dissolved oxygen (for instance) in the environment, experienced by microorganisms at the industrial scale. Oscillations in dissolved oxygen (DO) concentration are not uncommon. Furthermore, these fluctuations can be exacerbated with poor mixing and mass transfer limitations, especially in fermentations with filamentous fungus as the microbial cell factory. In this work, the response of glucose-limited chemostat cultures of an industrial Penicillium chrysogenum strain to different dissolved oxygen levels was assessed under both DO shift-down (60% → 20%, 10% and 5%) and DO ramp-down (60% → 0% in 24 h) conditions. Collectively, the results revealed that the penicillin productivity decreased as the DO level dropped down below 20%, while the byproducts, e.g., 6-oxopiperidine-2-carboxylic acid (OPC) and 6-aminopenicillanic acid (6APA), accumulated. Following DO ramp-down, penicillin productivity under DO shift-up experiments returned to its maximum value in 60 h when the DO was reset to 60%. The result showed that a higher cytosolic redox status, indicated by NADH/NAD+, was observed in the presence of insufficient oxygen supply. Consistent with this, flux balance analysis indicated that the flux through the glyoxylate shunt was increased by a factor of 50 at a DO value of 5% compared to the reference control, favoring the maintenance of redox status. Interestingly, it was observed that, in comparison with the reference control, the penicillin productivity was reduced by 25% at a DO value of 5% under steady state conditions. Only a 14% reduction in penicillin productivity was observed as the DO level was ramped down to 0. Furthermore, intracellular levels of amino acids were less sensitive to DO levels at DO shift-down relative to DO ramp-down conditions; this difference could be caused by different timescales between turnover rates of amino acid pools (tens of seconds to minutes) and DO switches (hours to days at steady state and minutes to hours at ramp-down). In summary, this study showed that changes in oxygen availability can lead to rapid metabolite, flux and productivity responses, and dynamic DO perturbations could provide insight into understanding of metabolic responses in large-scale bioreactors.

Optimizing a High Performing Multiplex-CRISPRi P. putida strain with Integrated Metabolomics and 13C-Metabolic Flux Analyses

Microbial cell factory development often faces bottlenecks after initial rounds of design-build-test-learn (DBTL) cycles as engineered producers respond unpredictably to further genetic modifications. Thus, deciphering metabolic flux and correcting bottlenecks are key components of DBTL cycles. Here, a 14-gene edited Pseudomonas putida KT2440 strain for heterologous indigoidine production was examined using both 13C-metabolic flux analysis (13C-MFA) and metabolite measurements. The results indicated the conservation of the cyclic Entner-Doudoroff (ED)-EMP pathway flux, downregulation of the TCA cycle and pyruvate shunt, and glyoxylate shunt activation. At the metabolite level, the CRISPR/dCpf1-interference mediated multiplex repression decreased gluconate/2-ketogluconate secretion and altered several intracellular TCA metabolite concentrations, leading to succinate overflow. Further strain engineering based on the metabolic knowledge first employed an optimal ribosome binding site (RBS) to achieve stronger product-substrate growth coupling (1.6-fold increase). Then, deletion strains were constructed using ssDNA recombineering. Of the five strains tested, deletion of the PHA operon (ΔphaAZC-IID) resulted in a 2.2-fold increase in growth phase production compared to the optimal RBS construct. After 72 h of batch cultivation, the ΔphaAZC-IID strain had 1.5-fold and 1.8-fold increases of indigoidine titer compared to the improved RBS construct and the original strain, respectively. Overall, the findings provided actionable DBTL targets as well as insights into physiological responses and flux buffering when new recombineering tools were used for engineering P. putida KT2440.

Functional analysis of H + -pumping membrane-bound pyrophosphatase, ADP-glucose synthase, and pyruvate phosphate dikinase as pyrophosphate sources in Clostridium thermocellum

The atypical glycolysis of Clostridium thermocellum is characterized by the use of pyrophosphate (PP i ) as phosphoryl donor for phosphofructokinase (Pfk) and pyruvate phosphate dikinase (Ppdk) reactions. Previously, biosynthetic PP i was calculated to be stoichiometrically insufficient to drive glycolysis. This study investigates the role of a H + -pumping membrane-bound pyrophosphatase, glycogen cycling, a predicted Ppdk–malate shunt cycle and acetate cycling in generating PP i . Knockout studies and enzyme assays confirmed that clo1313_0823 encodes a membrane-bound pyrophosphatase. Additionally, clo1313_0717-0718 was confirmed to encode ADP-glucose synthase by knockouts, glycogen measurements in C. thermocellum and heterologous expression in E. coli . Unexpectedly, individually-targeted gene deletions of the four putative PP i sources did not have a significant phenotypic effect. Although combinatorial deletion of all four putative PP i sources reduced the growth rate by 22% (0.30±0.01 h −1 ) and the biomass yield by 38% (0.18±0.00 g biomass g substrate −1 ), this change was much smaller than what would be expected for stoichiometrically essential PP i -supplying mechanisms. Growth-arrested cells of the quadruple knockout readily fermented cellobiose indicating that the unknown PP i -supplying mechanisms are independent of biosynthesis. An alternative hypothesis that ATP-dependent Pfk activity circumvents a need for PP i altogether, was falsified by enzyme assays, heterologous expression of candidate genes and whole-genome sequencing. As a secondary outcome, enzymatic assays confirmed functional annotation of clo1313_1832 as ATP- and GTP-dependent fructokinase. These results indicate that the four investigated PP i sources individually and combined play no significant PP i -supplying role and the true source(s) of PP i , or alternative phosphorylating mechanisms, that drive glycolysis in C. thermocellum remain(s) elusive. IMPORTANCE Increased understanding of the central metabolism of C. thermocellum is important from a fundamental as well as from a sustainability and industrial perspective. In addition to showing that H + -pumping membrane-bound PPase, glycogen cycling, a Ppdk–malate shunt cycle, and acetate cycling are not significant sources of PP i supply, this study adds functional annotation of four genes and availability of an updated PP i stoichiometry from biosynthesis to the scientific domain. Together, this aids future metabolic engineering attempts aimed to improve C. thermocellum as a cell factory for sustainable and efficient production of ethanol from lignocellulosic material through consolidated bioprocessing with minimal pretreatment. Getting closer to elucidating the elusive source of PP i , or alternative phosphorylating mechanisms, for the atypical glycolysis is itself of fundamental importance. Additionally, the findings of this study directly contribute to investigations into trade-offs between thermodynamic driving force versus energy yield of PP i - and ATP-dependent glycolysis.

Development of an Efficient Gene Editing Tool in Schizochytrium sp. and Improving Its Lipid and Terpenoid Biosynthesis

Schizochytrium sp. HX-308 is a marine microalga with fast growth and high lipid content, which has potential as microbial cell factories for lipid compound biosynthesis. It is significant to develop efficient genetic editing tool and discover molecular target in Schizochytrium sp. HX-308 for lipid compound biosynthesis. In this study, we developed an efficient gene editing tool in HX-308 which was mediated by Agrobacterium tumefaciens AGL-1. Results showed that the random integration efficiency reached 100%, and the homologous recombination efficiency reached about 30%. Furthermore, the metabolic pathway of lipid and terpenoid biosynthesis were engineered. Firstly, the acetyl-CoA c-acetyltransferase was overexpressed in HX-308 with a strong constitutive promoter. With the overexpression of acetyl-CoA c-acetyltransferase, more acetyl-CoA was used to synthesize terpenoids, and the production of squalene, β-carotene and astaxanthin was increased 5.4, 1.8, and 2.4 times, respectively. Interestingly, the production of saturated fatty acids and polyunsaturated fatty acids also changed. Moreover, three Acyl-CoA oxidase genes which catalyze the first step of β-oxidation were knocked out using homologous recombination. Results showed that the production of lipids increased in the three knock-out strains. Our results demonstrated that the A. tumefaciens-mediated transformation method will be of great use for the study of function genes, as well as developing Schizochytrium sp. as a strong cell factory for producing high value products.

Characterization of Clay from Isan Ekiti Deposits for Making the Ceramic Water Purifier

Introduction: Despite the efforts of Governments and non-governmental organizations in sponsoring ceramic water purifier (CWP) filter project across the globe, some rural communities in Ekiti State, Nigeria are yet to benefit from it. One of the major technical difficulties hindering the making of CWP filter cells in many places was the adaptation to the sand, clay and sawdust mixture necessary to obtain the correct filter properties. Aim: In this study, some salient properties of clay from orudi, arade and oturo deposits at Isan Ekiti were assessed in the light of their usefulness for making the ceramic water filter cells. Methodology: The particle size distribution analysis and consistency tests were carried out on the crude clay. Also the shrinkage, effects of percentage constituents of burnout on porosity, filtration rate and quality of filtrate were measured, using standard methods. Results: Results show that arade has the appropriate technical properties for making the filter cell. The chosen clay sample has its deposit less than 2 kilometers to the point of making. About 56% of its particles are less than 0.075mm equivalent spherical diameter (ESD). A composition of ratio 50:50 by volume mixture of clay to sawdust has the porosity of 54. 55% while that of ratio 40:60 is 55.56%. Samples made of these batches were capable of reducing the water turbidity Neflometric Turbidity Unit (NTU) of contaminated water by 95%. The 50:50 samples have the higher capacity of reducing total coliform count by 50.28% as against 25.07% for 40:60 samples. Conclusion: Given appropriate facilities and training, with the abundant clay at arade deposit, the pottery center is a place where CWP filter cell factory could be established.

Establishment of Kluyveromyces marxianus as a Microbial Cell Factory for Lignocellulosic Processes: Production of High Value Furan Derivatives

The establishment of lignocellulosic biorefineries is dependent on microorganisms being able to cope with the stressful conditions resulting from the release of inhibitory compounds during biomass processing. The yeast Kluyveromyces marxianus has been explored as an alternative microbial factory due to its thermotolerance and ability to natively metabolize xylose. The lignocellulose-derived inhibitors furfural and 5-hydroxymethylfurfural (HMF) are considered promising building-block platforms that can be converted into a wide variety of high-value derivatives. Here, several K. marxianus strains, isolated from cocoa fermentation, were evaluated for xylose consumption and tolerance towards acetic acid, furfural, and HMF. The potential of this yeast to reduce furfural and HMF at high inhibitory loads was disclosed and characterized. Our results associated HMF reduction with NADPH while furfural-reducing activity was higher with NADH. In addition, furans’ inhibitory effect was higher when combined with xylose consumption. The furan derivatives produced by K. marxianus in different conditions were identified. Furthermore, one selected isolate was efficiently used as a whole-cell biocatalyst to convert furfural and HMF into their derivatives, furfuryl alcohol and 2,5-bis(hydroxymethyl)furan (BHMF), with high yields and productivities. These results validate K. marxianus as a promising microbial platform in lignocellulosic biorefineries.