Journal of Industrial Microbiology & Biotechnology最新文献

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%. Y. 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 h, the mutant Y. lipolytica 5-14-E6 erythritol yield reached 178 g/L.

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.

The yeast Komagataella phaffii has become a popular host strain among biotechnology startup companies for producing recombinant proteins for food and adult nutrition applications. K. phaffii is a host of choice due to its long history of safe use, open access to protocols and strains, a secretome free of host proteins and proteases, and contract manufacturing organizations with deep knowledge in bioprocess scale-up. However, a recent publication highlighted the abundance of an unknown polysaccharide that accumulates in the supernatant during fermentation. This poses a significant challenge in using K. phaffii as a production host. This polysaccharide leads to difficulties in achieving high purity products and requires specialized and costly downstream processing steps for removal. In this study, we describe the use of the common K. phaffii host strain YB-4290 for production of the bioactive milk protein lactoferrin. Upon purification of lactoferrin using membrane-based separation methods, significant amounts of carbohydrate were co-purified with the protein. It was determined that the carbohydrate is mostly composed of mannose residues with minor amounts of glucose and glucosamine. The polysaccharide fraction has an average molecular weight of 50 kDa and consists mainly of mannan, galactomannan and amylose. In addition, a large fraction of the carbohydrate has an unknown structure likely composed of oligosaccharides. Additional strains were tested in fermentation to further understand the source of the carbohydrates. The commonly used industrial hosts, BG10 and YB-4290, produce a basal level of exopolysaccharide; YB-4290 producing slightly more than BG10. Overexpression of recombinant protein stimulates exopolysaccharide production well above levels produced by the host strains alone. Overall, this study aims to provide a foundation for developing methods to improve the economics of recombinant protein production using K. phaffii as a production host.

Fixed nitrogen fertilizers feed fifty percent 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 nitrogen (N2) into ammonia (NH3) using methane (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 ammonia (NH3) that could ultimately be developed to support compact, remote, ammonia (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 methane (CH4) molecules to L-lactate (C3H6O3) and powered gaseous nitrogen (N2) conversion to ammonia (NH3). The design used division of labor and mutualistic metabolism strategies to address the oxygen sensitivity of nitrogenase and maximize methane oxidation efficiency. Media pH and salinity were central variables supporting co-cultivation. Carbon concentration heavily influenced ammonia production. Smaller scale ammonia (NH3) production near dispersed, abundant, and renewable methane (CH4) sources could reduce disruption risks and capitalize on untapped energy resources.

To develop a host-vector system for use in thermophilic Streptomyces, multi-copy plasmids were screened for thermophilic Streptomyces species using data from public bioresource centers (JCM and NBRC). Of 27 thermophilic Streptomyces strains, three harbored plasmids. One plasmid (pSTVU1), derived from S. thermovulgaris NBRC 16615 (= JCM 4520, ATCC 19284, DSM 40444, ISP 5444, NRRL B-12375, NCIMB 10078), was multi-copy and relatively small in size. Analysis of the sequence of this multi-copy plasmid revealed that it was 7 838 bp and contained at least 10 predicted open reading frames (ORFs). The plasmid was introduced into 14 thermophilic Streptomyces strains (of 18 strains examined) and several mesophilic Streptomyces strains (S. lividans, S. parvulus, and S. avermitilis). pSTVU1 can be transferred by mixed culture because the plasmid encodes the ORF that regulates the transfer function. Plasmid transfer was observed not only between strains within the same species but also between mesophilic Streptomyces and thermophilic Streptomyces (and vice versa); however, the efficiency of this transfer was extremely low. We also confirmed that a derivative of pSTVU1 can be used as a multi-copy vector in the gene expression system that is expected to exhibit gene-dosage effects, establishing a method for efficient production of thermophilic α-amylase.

Driven by demand for more sustainable products, research and capital investment has been committed to developing microbially produced oils. While researchers have shown oleaginous yeasts and other microbes can produce low-carbon footprint oils by leveraging waste streams as energy sources, previous analyses have not fully explored the quantity of available waste streams and in turn economy-of-scale enabled on capital and operating expenses. This paper makes parallels to 2G ethanol facilities, enabling a data-driven understanding of large-scale production economics. Production costs are broken down for a variety of scenarios. The analysis finds that reaching price parity with large-scale commodity oils (e.g., palm oil, high-oleic cooking oils, biofuels feedstock oils, lauric acid) is not possible today and unlikely even under aggressive future assumptions about strain productivity. Instead, commercial production must be targeted at end markets where sustainability-conscious consumers are willing to pay the price premiums identified in this paper.