Journal of Industrial Microbiology & Biotechnology最新文献

To determine the performance of a sophorolipid biosurfactant production process, it is important to have accurate and specific analytical techniques in place. Among the most popular are the anthrone assay, gravimetric quantification (hexane:ethyl acetate extraction), and high-performance liquid chromatography (HPLC). The choice of analytical tool varies depending on cost, availability, and ease of use; however, these techniques have never been compared directly against one another. In this work, 75 fermentation broths with varying product/substrate concentrations were comprehensively tested with the 3 techniques and compared. HPLC-ultraviolet detection (198 nm) was capable of quantifying C18:1 subterminal hydroxyl diacetylated lactonic sophorolipid down to a lower limit of 0.3 g/L with low variability (<3.21%). Gravimetric quantification of the broths following liquid:liquid extraction with hexane and ethyl acetate showed some linearity (R2 = .658) when compared to HPLC but could not quantify lower than 11.06 g/L, even when no sophorolipids were detected in the sample, highlighting the non-specificity of the method to co-extract non-sophorolipid components in the final gravimetric measure. The anthrone assay showed no linearity (R2 = .129) and was found to cross-react with media components (rapeseed oil, corn steep liquor, glucose), leading to consistent overestimation of sophorolipid concentration. The appearance of poor biomass separation during sample preparation with centrifugation was noted and resolved with a novel sample preparation method with pure ethanol. Extensive analysis and comparisons of the most common sophorolipid quantification techniques are explored and the limitations/advantages are highlighted. The findings provide a guide for scientists to make an informed decision on the suitable quantification tool that meets their needs, exploring all aspects of the analysis process from harvest, sample preparation, and analysis.

Yarrowia lipolytica is widely used for the industrial production of the natural sweetener erythritol. Despite improvements in fermentation process control and metabolic pathway regulation, bottlenecks still exist in terms of yield and screening technology. Therefore, we constructed an artificial sensor system for effective erythritol detection, established a single-cell droplet-based high-throughput screening system based on fluorescence-activated cell sorting, and obtained Y. lipolytica with improved erythritol production through mutagenesis and high-throughput screening. We used a droplet generator to co-cultivate Y. lipolytica 5-14 with Escherichia coli and used the E. coli fluorescent signal to detect the concentration of erythritol synthesized by Y. lipolytica 5-14 for high-throughput screening. Strains were subjected to UV mutagenesis for 120 s. Under optimized fermentation conditions using Y. lipolytica mutants in 96-well plates, the screening efficiency reached 16.7%. Yarrowia lipolytica 5-14-E6 showed a 21% increase in erythritol to 109.84 g/L. After fermentation at 30°C in a 100-m3 fermenter for 75 hr, the mutant Y. lipolytica 5-14-E6 erythritol yield reached 178 g/L.

One-sentence summary: In this study, we constructed an artificial sensor system for effective erythritol detection, established a single-cell droplet-based high-throughput screening system based on fluorescence-activated cell sorting, and induced and screened mutant strains of Yarrowia lipolytica with improved erythritol production through mutagenesis and high-throughput screening.

Pyrroloquinoline quinone (PQQ) is one of the important coenzymes in living organisms. In acetic acid bacteria (AAB), it plays a crucial role in the alcohol respiratory chain, as a coenzyme of alcohol dehydrogenase (ADH). In this work, the PQQ biosynthetic genes were overexpressed in Acetobacter pasteurianus CGMCC 3089 to improve the fermentation performance. The result shows that the intracellular and extracellular PQQ contents in the recombinant strain A. pasteurianus (pBBR1-p264-pqq) were 152.53% and 141.08% higher than those of the control A. pasteurianus (pBBR1-p264), respectively. The catalytic activity of ADH and aldehyde dehydrogenase increased by 52.92% and 67.04%, respectively. The results indicated that the energy charge and intracellular ATP were also improved in the recombinant strain. The acetic acid fermentation was carried out using a 5 L self-aspirating fermenter, and the acetic acid production rate of the recombinant strain was 23.20% higher compared with the control. Furthermore, the relationship between the PQQ and acetic acid tolerance of cells was analyzed. The biomass of recombinant strain was 180.2%, 44.3%, and 38.6% higher than those of control under 2%, 3%, and 4% acetic acid stress, respectively. After being treated with 6% acetic acid for 40 min, the survival rate of the recombinant strain was increased by 76.20% compared with the control. Those results demonstrated that overexpression of PQQ biosynthetic genes increased the content of PQQ, therefore improving the acetic acid fermentation and the cell tolerance against acetic acid by improving the alcohol respiratory chain and energy metabolism.

One sentence summary: The increase in PQQ content enhances the activity of the alcohol respiratory chain of Acetobacter pasteurianus, and the increase in energy charge enhances the tolerance of cells against acetic acid, therefore, improving the efficiency of acetic acid fermentation.

Prediction and process monitoring during natural attenuation, bioremediation, and biotreatment require effective strategies for detection and enumeration of the responsible bacteria. The use of 2,4-dinitroanisole (DNAN) as a component of insensitive munitions leads to environmental contamination of firing ranges and manufacturing waste streams. Nocardioides sp. strain JS1661 degrades DNAN under aerobic conditions via a pathway involving an unusual DNAN demethylase. We used the deeply branched sequences of DNAN degradation functional genes as a target for development of a molecular method for detection of the bacteria. A qPCR assay was designed for the junction between dnhA and dnhB, the adjacent genes encoding DNAN demethylase. The assay allowed reproducible enumeration of JS1661 during growth in liquid media and soil slurries. Results were consistent with biodegradation of DNAN, accumulation of products, and classical biomass estimates, including most probable number and OD600. The results provide a sensitive and specific molecular method for prediction of degradation potential and process evaluation during degradation of DNAN.

One-sentence summary: A unique target sequence in functional genes enables the design of a simple and specific qPCR assay for enumeration of aerobic 2,4-dinitroanisole-degrading bacteria in soil and water.

Automation of metabolite control in fermenters is fundamental to develop vaccine manufacturing processes more quickly and robustly. We created an end-to-end process analytical technology and quality by design-focused process by replacing manual control of metabolites during the development of fed-batch bioprocesses with a system that is highly adaptable and automation-enabled. Mid-infrared spectroscopy with an attenuated total reflectance probe in-line, and simple linear regression using the Beer-Lambert Law, were developed to quantitate key metabolites (glucose and glutamate) from spectral data that measured complex media during fermentation. This data was digitally connected to a process information management system, to enable continuous control of feed pumps with proportional-integral-derivative controllers that maintained nutrient levels throughout fed-batch stirred-tank fermenter processes. Continuous metabolite data from mid-infrared spectra of cultures in stirred-tank reactors enabled feedback loops and control of the feed pumps in pharmaceutical development laboratories. This improved process control of nutrient levels by 20-fold and the drug substance yield by an order of magnitude. Furthermore, the method is adaptable to other systems and enables soft sensing, such as the consumption rate of metabolites. The ability to develop quantitative metabolite templates quickly and simply for changing bioprocesses was instrumental for project acceleration and heightened process control and automation.

One-sentence summary: Intelligent digital control systems using continuous in-line metabolite data enabled end-to-end automation of fed-batch processes in stirred-tank reactors.

With the expansion of domesticated microbes producing biomaterials and chemicals to support a growing circular bioeconomy, the variety of waste and sustainable substrates that can support microbial growth and production will also continue to expand. The diversity of these microbes also requires a range of compatible genetic tools to engineer improved robustness and economic viability. As we still do not fully understand the function of many genes in even highly studied model microbes, engineering improved microbial performance requires introducing genome-scale genetic modifications followed by screening or selecting mutants that enhance growth under prohibitive conditions encountered during production. These approaches include adaptive laboratory evolution, random or directed mutagenesis, transposon-mediated gene disruption, or CRISPR interference (CRISPRi). Although any of these approaches may be applicable for identifying engineering targets, here we focus on using CRISPRi to reduce the time required to engineer more robust microbes for industrial applications.

One-sentence summary: The development of genome scale CRISPR-based libraries in new microbes enables discovery of genetic factors linked to desired traits for engineering more robust microbial systems.

Fixed nitrogen fertilizers feed 50% of the global population, but most fixed nitrogen production occurs using energy-intensive Haber-Bosch-based chemistry combining nitrogen (N2) from air with gaseous hydrogen (H2) from methane (CH4) at high temperatures and pressures in large-scale facilities sensitive to supply chain disruptions. This work demonstrates the biological transformation of atmospheric N2 into ammonia (NH3) using CH4 as the sole carbon and energy source in a single vessel at ambient pressure and temperature, representing a biological "room-pressure and room-temperature" route to NH3 that could ultimately be developed to support compact, remote, NH3 production facilities amenable to distributed biomanufacturing. The synthetic microbial co-culture of engineered methanotroph Methylomicrobium buryatense (now Methylotuvimicrobium buryatense) and diazotroph Azotobacter vinelandii converted three CH4 molecules to l-lactate (C3H6O3) and powered gaseous N2 conversion to NH3. The design used division of labor and mutualistic metabolism strategies to address the oxygen sensitivity of nitrogenase and maximize CH4 oxidation efficiency. Media pH and salinity were central variables supporting co-cultivation. Carbon concentration heavily influenced NH3 production. Smaller-scale NH3 production near dispersed, abundant, and renewable CH4 sources could reduce disruption risks and capitalize on untapped energy resources.

One-sentence summary: Co-culture of engineered microorganisms Methylomicrobium buryatense and Azotobacter vinelandii facilitated the use of methane gas as a sole carbon feedstock to produce ammonia in an ambient temperature, atmospheric pressure, single-vessel system.

Cyclophilin B (CypB), a significant member of immunophilins family with peptidyl-prolyl cis-trans isomerase (PPIase) activity, is crucial for the growth and metabolism of prokaryotes and eukaryotes. Sporothrix globosa (S. globosa), a principal pathogen in the Sporothrix complex, causes sporotrichosis. Transcriptomic analysis identified the cypB gene as highly expressed in S. globosa. Our previous study demonstrated that the recombinant Escherichia coli strain containing SgcypB gene failed to produce sufficient product when it was induced to express the protein, implying the potential toxicity of recombinant protein to the bacterial host. Bioinformatics analysis revealed that SgCypB contains transmembrane peptides within the 52 amino acid residues at the N-terminus and 21 amino acids near the C-terminus, and 18 amino acid residues within the cytoplasm. AlphaFold2 predicted a SgCypB 3D structure in which there is an independent PPIase domain consisting of a spherical extracellular part. Hence, we chose to express the extracellular domain to yield high-level recombinant protein with PPIase activity. Finally, we successfully produced high-yield, truncated recombinant CypB protein from S. globosa (SgtrCypB) that retained characteristic PPIase activity without host bacterium toxicity. This study presents an alternative expression strategy for proteins toxic to prokaryotes, such as SgCypB.

One-sentence summary: The recombinant cyclophilin B protein of Sporothrix globosa was expressed successfully by retaining extracellular domain with peptidyl-prolyl cis-trans isomerase activity to avoid toxicity to the host bacterium.

Chain elongating bacteria are a unique guild of strictly anaerobic bacteria that have garnered interest for sustainable chemical manufacturing from carbon-rich wet and gaseous waste streams. They produce C6-C8 medium-chain fatty acids, which are valuable platform chemicals that can be used directly, or derivatized to service a wide range of chemical industries. However, the application of chain elongating bacteria for synthesizing products beyond C6-C8 medium-chain fatty acids has not been evaluated. In this study, we assess the feasibility of expanding the product spectrum of chain elongating bacteria to C9-C12 fatty acids, along with the synthesis of C6 fatty alcohols, dicarboxylic acids, diols, and methyl ketones. We propose several metabolic engineering strategies to accomplish these conversions in chain elongating bacteria and utilize constraint-based metabolic modelling to predict pathway stoichiometries, assess thermodynamic feasibility, and estimate ATP and product yields. We also evaluate how producing alternative products impacts the growth rate of chain elongating bacteria via resource allocation modelling, revealing a trade-off between product chain length and class versus cell growth rate. Together, these results highlight the potential for using chain elongating bacteria as a platform for diverse oleochemical biomanufacturing and offer a starting point for guiding future metabolic engineering efforts aimed at expanding their product range.

One-sentence summary: In this work, the authors use constraint-based metabolic modelling and enzyme cost minimization to assess the feasibility of using metabolic engineering to expand the product spectrum of anaerobic chain elongating bacteria.

Microbial conversion of lignocellulosic biomass represents an alternative route for production of biofuels and bioproducts. While researchers have mostly focused on engineering strains such as Rhodotorula toruloides for better bisabolene production as a sustainable aviation fuel, less is known about the impact of the feedstock heterogeneity on bisabolene production. Critical material attributes like feedstock composition, nutritional content, and inhibitory compounds can all influence bioconversion. Further, the given feedstocks can have a marked influence on selection of suitable pretreatment and hydrolysis technologies, optimizing the fermentation conditions, and possibly even modifying the microorganism's metabolic pathways, to better utilize the available feedstock. This work aimed to examine and understand how variations in corn stover batches, anatomical fractions, and storage conditions impact the efficiency of bisabolene production by R. toruloides. All of these represent different facets of feedstock heterogeneity. Deacetylation, mechanical refining, and enzymatic hydrolysis of these variable feedstocks served as the basis of this research. The resulting hydrolysates were converted to bisabolene via fermentation, a sustainable aviation fuel precursor, using an engineered R. toruloides strain. This study showed that different sources of feedstock heterogeneity can influence microbial growth and product titer in counterintuitive ways, as revealed through global analysis of protein expression. The maximum bisabolene produced by R. toruloides was on the stalk fraction of corn stover hydrolysate (8.89 ± 0.47 g/L). Further, proteomics analysis comparing the protein expression between the anatomic fractions showed that proteins relating to carbohydrate metabolism, energy production, and conversion as well as inorganic ion transport metabolism were either significantly upregulated or downregulated. Specifically, downregulation of proteins related to the iron-sulfur cluster in stalk fraction suggests a coordinated response by R. toruloides to maintain overall metabolic balance, and this was corroborated by the concentration of iron in the feedstocks.

One-sentence summary: This study elucidates the effects of different sources of corn stover on bisabolene production by engineered Rhodotorula toruloides, highlighting the importance of understanding feedstock variability to enhance bioprocess efficiency and economic outcomes.