Journal of Industrial Microbiology & Biotechnology最新文献

Effective microbial bioprocessing relies on maintaining ideal cultivation conditions, highlighting the necessity for tools that monitor and regulate cellular performance and robustness. This study evaluates a fed-batch cultivation control system based on at-line flow cytometry monitoring of intact yeast cells having a fluorescent transcription factor-based redox biosensor. Specifically, the biosensor assesses the response of an industrial xylose-fermenting Saccharomyces cerevisiae strain carrying the TRX2p-yEGFP biosensor for NADPH/NADP+ ratio imbalance when exposed to furfural. The developed control system successfully detected biosensor output and automatically adjusted furfural feed rate, ensuring physiological fitness at high furfural levels. Moreover, the single-cell measurements enabled the monitoring of subpopulation dynamics, enhancing control precision over traditional methods. The presented automated control system highlights the potential of combining biosensors and flow cytometry for robust microbial cultivations by leveraging intracellular properties as control inputs.

One-sentence summary: An automated control system using flow cytometry and biosensors enhances microbial bioprocessing by regulating cellular performance in response to the environmental stressor furfural.

Despite their prevalent use in drug discovery and protein biochemistry, non-canonical amino acids are still challenging to synthesize through purely chemical means. In recent years, biocatalysis has emerged as a transformative paradigm for small-molecule synthesis. One strategy to further empower biocatalysis is to use it in combination with modern chemical reactions and take advantage of the strengths of each method to enable access to challenging structural motifs that were previously unattainable using each method alone. In this Mini-Review, we highlight several recent case studies that feature the synergistic use of chemical and enzymatic transformations in one pot to synthesize novel non-canonical amino acids.

One-sentence summary: This Mini-Review highlights several recent case studies that feature the synergistic use of chemical and enzymatic transformations in one pot to synthesize novel non-canonical amino acids.

Legionella is a bacterial genus found in natural aquatic environments, as well as domestic and industrial water systems. Legionella presents potential human health risks when aerosolized and inhaled by at-risk individuals and is commonly monitored at locations with likelihood of proliferation and human exposure. Legionella monitoring is widely performed using culture-based testing, which faces limitations including turnaround time and interferences. Molecular biology methodologies, including quantitative polymerase chain reaction (qPCR), are being explored to supplement or replace culture-based testing because of faster turnaround and lower detection limits, allowing for more rapid water remediation measures. In this study, three methods were compared by testing industrial water samples: culture-based testing by a certified lab, high throughput qPCR testing (HT qPCR), and field deployable low throughput qPCR testing (LT qPCR). The qPCR test methods reported more positive results than culture testing, indicating improved sensitivity and specificity. The LT qPCR test is portable with quick turnaround times, and can be leveraged for environmental surveillance, process optimization, monitoring, and onsite case investigations. The LT qPCR test had high negative predictive value and would be a useful tool for negative screening of Legionella samples from high-risk environments and/or outbreak investigations to streamline samples for culture testing.

One-sentence summary: This study compared three test methods for Legionella to evaluate performance of a low throughput quantitative polymerase chain reaction (LT qPCR) test for Legionella that can be used onsite; the study found that the high throughput (HT) and LT qPCR tests used in this study gave more positive results than culture testing, and the results indicated a similar negative predictive value for the HT and LT qPCR tests, supporting that the LT qPCR method could be useful for negative screening of Legionella samples in industrial water systems onsite.

The Gram-negative betaproteobacterium Cupriavidus necator is a chemolithotroph that can convert carbon dioxide into biomass. Cupriavidus necator has been engineered to produce a variety of high-value chemicals in the past. However, there is still a lack of a well-characterized toolbox for gene expression and genome engineering. Development and optimization of biosynthetic pathways in metabolically engineered microorganisms necessitates control of gene expression via functional genetic elements such as promoters, ribosome binding sites (RBSs), and codon optimization. In this work, a set of inducible and constitutive promoters were validated and characterized in C. necator, and a library of RBSs was designed and tested to show a 50-fold range of expression for green fluorescent protein (gfp). The effect of codon optimization on gene expression in C. necator was studied by expressing gfp and mCherry genes with varied codon-adaptation indices and was validated by expressing codon-optimized variants of a C12-specific fatty acid thioesterase to produce dodecanoic acid. We discuss further hurdles that will need to be overcome for C. necator to be widely used for biosynthetic processes.

The industrial amino acid production workhorse, Corynebacterium glutamicum naturally produces low levels of 2,3,5,6-tetramethylpyrazine (TMP), a valuable flavor, fragrance, and commodity chemical. Here, we demonstrate TMP production (∼0.8 g L-1) in C. glutamicum type strain ATCC13032 via overexpression of acetolactate synthase and/or α-acetolactate decarboxylase from Lactococcus lactis in CGXII minimal medium supplemented with 40 g L-1 glucose. This engineered strain also demonstrated growth and TMP production when the minimal medium was supplemented with up to 40% (v v-1) hydrolysates derived from ionic liquid-pretreated sorghum biomass. A key objective was to take the fully engineered strain developed in this study and interrogate medium parameters that influence the production of TMP, a critical post-strain engineering optimization. Design of experiments in a high-throughput plate format identified glucose, urea, and their ratio as significant components affecting TMP production. These two components were further optimized using response surface methodology. In the optimized CGXII medium, the engineered strain could produce up to 3.56 g L-1 TMP (4-fold enhancement in titers and 2-fold enhancement in yield, mol mol-1) from 80 g L-1 glucose and 11.9 g L-1 urea in shake flask batch cultivation.

One-sentence summary: Corynebacterium glutamicum was metabolically engineered to produce 2,3,5,6-tetramethylpyrazine followed by a design of experiments approach to optimize medium components for high-titer production.

Out of the 166 articles published in Journal of Industrial Microbiology and Biotechnology (JIMB) in 2019-2020 (not including special issues or review articles), 51 of them used a statistical test to compare two or more means. The most popular test was the (Standard) t-test, which often was used to compare several pairs of means. Other statistical procedures used included Fisher's least significant difference (LSD), Tukey's honest significant difference (HSD), and Welch's t-test; and to a lesser extent Bonferroni, Duncan's Multiple Range, Student-Newman-Keuls, and Kruskal-Wallis tests. This manuscript examines the performance of some of these tests with simulated experimental data, typical of those reported by JIMB authors. The results show that many of the most common procedures used by JIMB authors result in statistical conclusions that are prone to have large false positive (Type I) errors. These error-prone procedures included the multiple t-test, multiple Welch's t-test, and Fisher's LSD. These multiple comparisons procedures were compared with alternatives (Fisher-Hayter, Tukey's HSD, Bonferroni, and Dunnett's t-test) that were able to better control Type I errors.

Non-technical summary: The aim of this work was to review and recommend statistical procedures for Journal of Industrial Microbiology and Biotechnology authors who often compare the effect of several treatments on microorganisms and their functions.

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, 3 harbored plasmids. One plasmid (pSTVU1), derived from S. thermovulgaris NBRC 16615 (= JCM 4520, ATCC 19284, DSM 40444, ISP 5444, NRRL B-12375, and 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. 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.

One-sentence summary: A multi-copy plasmid was identified in thermophilic Streptomyces and used to develop a gene cloning system for thermophilic Streptomyces species.

Agricultural waste valorisation provides a sustainable solution to waste management, and combining waste utilisation with commodity production allows for responsible production processes. Recombinant Aspergillus niger D15 strains expressing fungal endoglucanases (Trichoderma reesei eg1 and eg2 and Aspergillus carneus aceg) were evaluated for their ability to utilise lactose as a carbon source to determine whether dairy waste could be used as a feedstock for enzyme production. The recombinant A. niger D15[eg1]PyrG, D15[eg2]PyrG, and D15[aceg]PyrG strains produced maximum endoglucanase activities of 34, 54, and 34 U/mL, respectively, on lactose and 23, 27, and 22 U/mL, respectively, on whey. The A. niger D15[eg2]PyrG strain was used to optimise the whey medium. Maximum endoglucanase activity of 46 U/mL was produced on 10% whey medium containing 0.6% NaNO3. The results obtained indicate that dairy whey can be utilised as a feedstock for recombinant enzyme production. However, variations in enzyme activities were observed and require further investigation.

The demand for discovering novel microbial secondary metabolites is growing to address the limitations in bioactivities such as antibacterial, antifungal, anticancer, anthelmintic, and immunosuppressive functions. Among microbes, the genus Streptomyces holds particular significance for secondary metabolite discovery. Each Streptomyces species typically encodes approximately 30 secondary metabolite biosynthetic gene clusters (smBGCs) within its genome, which are mostly uncharacterized in terms of their products and bioactivities. The development of next-generation sequencing has enabled the identification of a large number of potent smBGCs for novel secondary metabolites that are imbalanced in number compared with discovered secondary metabolites. The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system has revolutionized the translation of enormous genomic potential into the discovery of secondary metabolites as the most efficient genetic engineering tool for Streptomyces. In this review, the current status of CRISPR/Cas applications in Streptomyces is summarized, with particular focus on the identification of secondary metabolite biosynthesis gene clusters and their potential applications.This review summarizes the broad range of CRISPR/Cas applications in Streptomyces for natural product discovery and production.

One-sentence summary: This review summarizes the broad range of CRISPR/Cas applications in Streptomyces for natural product discovery and production.

Corynebacterium glutamicum ATCC 13032 is a promising microbial chassis for industrial production of valuable compounds, including aromatic amino acids derived from the shikimate pathway. In this work, we developed two whole-cell, transcription factor based fluorescent biosensors to track cis,cis-muconic acid (ccMA) and chorismate in C. glutamicum. Chorismate is a key intermediate in the shikimate pathway from which value-added chemicals can be produced, and a shunt from the shikimate pathway can divert carbon to ccMA, a high value chemical. We transferred a ccMA-inducible transcription factor, CatM, from Acinetobacter baylyi ADP1 into C. glutamicum and screened a promoter library to isolate variants with high sensitivity and dynamic range to ccMA by providing benzoate, which is converted to ccMA intracellularly. The biosensor also detected exogenously supplied ccMA, suggesting the presence of a putative ccMA transporter in C. glutamicum, though the external ccMA concentration threshold to elicit a response was 100-fold higher than the concentration of benzoate required to do so through intracellular ccMA production. We then developed a chorismate biosensor, in which a chorismate inducible promoter regulated by natively expressed QsuR was optimized to exhibit a dose-dependent response to exogenously supplemented quinate (a chorismate precursor). A chorismate-pyruvate lyase encoding gene, ubiC, was introduced into C. glutamicum to lower the intracellular chorismate pool, which resulted in loss of dose dependence to quinate. Further, a knockout strain that blocked the conversion of quinate to chorismate also resulted in absence of dose dependence to quinate, validating that the chorismate biosensor is specific to intracellular chorismate pool. The ccMA and chorismate biosensors were dually inserted into C. glutamicum to simultaneously detect intracellularly produced chorismate and ccMA. Biosensors, such as those developed in this study, can be applied in C. glutamicum for multiplex sensing to expedite pathway design and optimization through metabolic engineering in this promising chassis organism.

One-sentence summary: High-throughput screening of promoter libraries in Corynebacterium glutamicum to establish transcription factor based biosensors for key metabolic intermediates in shikimate and β-ketoadipate pathways.