Journal of Industrial Microbiology & Biotechnology最新文献

The off-flavor of Pichia pastoris strains is a negative characteristic of proteins overexpressed with this yeast. In the present study, P. pastoris GS115 overexpressing an α-l-rhamnosidase was taken as the example to characterize the off-flavor via sensory evaluation, gas chromatography-mass spectrometer, gas chromatography-olfaction, and omission test. The result showed that the off-flavor was due to the strong sweaty note, and moderate metallic and plastic notes. Four volatile compounds, that is, tetramethylpyrazine, 2,4-di-tert-butylphenol, isovaleric acid, and 2-methylbutyric acid, were identified to be major contributors to the sweaty note. Dodecanol and 2-acetylbutyrolactone were identified to be contributors to the metallic and plastic notes, respectively. It is the first study on the off-flavor of P. pastoris strains, helping understand metabolites with off-flavor of this yeast. Interestingly, it is the first study illustrating 2-acetylbutyrolactone and dodecanol with plastic and metallic notes, providing new information about the aromatic contributors of biological products.

Importance: The methylotrophic yeast Pichia pastoris is an important host for the industrial expression of functional proteins. In our previous studies, P. pastoris strains have been sniffed with a strong off-flavor during the overexpression of various functional proteins, limiting the application of these proteins. Although many yeast strains have been reported with off-flavor, no attention has been paid to characterize the off-flavor in P. pastoris so far. Considering that P. pastoris has advantages over other established expression systems of functional proteins, it is of interest to identify the compounds with off-flavor synthesized in the overexpression of functional proteins with P. pastoris strains. In this study, the off-flavor synthesized from P. pastoris GS115 was characterized during the overexpression of an α-l-rhamnosidase, which helps understand the aromatic metabolites with off-flavor of P. pastoris strains. In addition, 2-acetylbutyrolactone and dodecanol were newly revealed with plastic and metallic notes, enriching the aromatic contributors of biological products. Thus, this study is important for understanding the metabolites with off-flavor of P. pastoris strains and other organisms, providing important knowledge to improve the flavor of products yielding with P. pastoris strains and other organisms.

One-sentence summary: Characterize the sensory and chemical profile of the off-flavor produced by one strain of P. pastoris in vitro.

The laccases from white-rot fungi exhibit high redox potential in treating phenolic compounds. However, their application in commercial purposes has been limited because of the relatively low productivity of the native hosts. Here, the laccase A-encoding gene lacA of Trametes sp. AH28-2 was overexpressed under the control of the strong promoter of cbh1 (Pcbh1), the gene encoding the endogenous cellobiohydrolase 1 (CBH1), in the industrial workhorse fungus Trichoderma reesei. Firstly, the lacA expression cassette was randomly integrated into the T. reesei chromosome by genetic transformation. The lacA gene was successfully transcribed, but the laccase couldn't be detected in the liquid fermentation condition. Meanwhile, it was found that the endoplasmic reticulum-associated degradation (ERAD) was strongly activated, indicating that the expression of LacA probably triggered intense endoplasmic reticulum (ER) stress. Subsequently, the lacA expression cassette was added with the downstream region of cbh1 (Tcbh1) to construct the new expression cassette lacA::Δcbh1, which could replace the cbh1 locus in the genome via homologous recombination. After genetic transformation, the lacA gene was integrated into the cbh1 locus and transcribed. And the unfolded protein response (UPR) and ERAD were only slightly induced, for which the loss of endogenous cellulase CBH1 released the pressure of secretion. Finally, the maximum laccase activity of 168.3 U/l was obtained in the fermentation broth. These results demonstrated that the reduction of secretion pressure by deletion of endogenous protein-encoding genes would be an efficient strategy for the secretion of heterologous target proteins in industrial fungi.

One-sentence summary: The reduction of the secretion pressure by deletion of the endogenous cbh1 gene can contribute to heterologous expression of the laccase (LacA) from Trametes sp. AH28-2 in Trichoderma reesei.

Tetrapyrroles represent a unique class of natural products that possess diverse chemical architectures and exhibit a broad range of biological functions. Accordingly, they attract keen attention from the natural product community. Many metal-chelating tetrapyrroles serve as enzyme cofactors essential for life, while certain organisms produce metal-free porphyrin metabolites with biological activities potentially beneficial for the producing organisms and for human use. The unique properties of tetrapyrrole natural products derive from their extensively modified and highly conjugated macrocyclic core structures. Most of these various tetrapyrrole natural products biosynthetically originate from a branching point precursor, uroporphyrinogen III, which contains propionate and acetate side chains on its macrocycle. Over the past few decades, many modification enzymes with unique catalytic activities, and the diverse enzymatic chemistries employed to cleave the propionate side chains from the macrocycles, have been identified. In this review, we highlight the tetrapyrrole biosynthetic enzymes required for the propionate side chain removal processes and discuss their various chemical mechanisms.

One-sentence summary: This mini-review describes various enzymes involved in the propionate side chain cleavages during the biosynthesis of tetrapyrrole cofactors and secondary metabolites.

Recent work in biosensors has shown promise to enable high throughput searches through large genetic libraries. However, just as physiological limitations and lack of in-depth mechanistic knowledge can prevent us from achieving high titers in microbial systems; similar roadblocks can appear in the application of biosensors. Here, we characterized a previously developed transcription-factor (ExuR) based galacturonate biosensor for its other cognate ligand, glucuronate. Though we saw an ideal response to glucuronate from the biosensor in controlled and ideal experimental circumstances, these results began to deviate from a well-behaved system when we explored the application of the sensor to different MIOX homologs. Through modifications to circuit architecture and culture conditions, we were able to decrease this variation and use these more optimal conditions to apply the biosensor for the separation of two closely related MIOX homologs.

One-sentence summary: In this work, a transcription-factor biosensor was investigated for its potential to screen a library of myo -inositol oxygenase variants while seeking to mitigate the impact the production pathway appeared to have on the biosensor.

Yield improvements in cell factories can potentially be obtained by fine-tuning the regulatory mechanisms for gene candidates. In pursuit of such candidates, we performed RNA-sequencing of two α-amylase producing Bacillus strains and predict hundreds of putative novel non-coding transcribed regions. Surprisingly, we found among hundreds of non-coding and structured RNA candidates that non-coding genomic regions are proportionally undergoing the highest changes in expression during fermentation. Since these classes of RNA are also understudied, we targeted the corresponding genomic regions with CRIPSRi knockdown to test for any potential impact on the yield. From differentially expression analysis, we selected 53 non-coding candidates. Although CRISPRi knockdowns target both the sense and the antisense strand, the CRISPRi experiment cannot link causes for yield changes to the sense or antisense disruption. Nevertheless, we observed on several instances with strong changes in enzyme yield. The knockdown targeting the genomic region for a putative antisense RNA of the 3' UTR of the skfA-skfH operon led to a 21% increase in yield. In contrast, the knockdown targeting the genomic regions of putative antisense RNAs of the cytochrome c oxidase subunit 1 (ctaD), the sigma factor sigH, and the uncharacterized gene yhfT decreased yields by 31 to 43%.

The biotechnological production of methyl ketones is a sustainable alternative to fossil-derived chemical production. To date, the best host for microbial production of methyl ketones is a genetically engineered Pseudomonas taiwanensis VLB120 ∆6 pProd strain, achieving yields of 101 mgg-1 on glucose in batch cultivations. For competitiveness with the petrochemical production pathway, however, higher yields are necessary. Co-feeding can improve the yield by fitting the carbon-to-energy ratio to the organism and the target product. In this work, we developed co-feeding strategies for P. taiwanensis VLB120 ∆6 pProd by combined metabolic modeling and experimental work. In a first step, we conducted flux balance analysis with an expanded genome-scale metabolic model of iJN1463 and found ethanol as the most promising among five cosubstrates. Next, we performed cultivations with ethanol and found the highest reported yield in batch production of methyl ketones with P. taiwanensis VLB120 to date, namely, 154 mg g-1 methyl ketones. However, ethanol is toxic to the cell, which reflects in a lower substrate consumption and lower product concentrations when compared to production on glucose. Hence, we propose cofeeding ethanol with glucose and find that, indeed, higher concentrations than in ethanol-fed cultivation (0.84 g Laq-1 with glucose and ethanol as opposed to 0.48 g Laq-1 with only ethanol) were achieved, with a yield of 85 mg g-1. In a last step, comparing experimental with computational results suggested the potential for improving the methyl ketone yield by fed-batch cultivation, in which cell growth and methyl ketone production are separated into two phases employing optimal ethanol to glucose ratios.

One-sentence summary: By combining computational and experimental work, we demonstrate that feeding ethanol in addition to glucose improves the yield of biotechnologically produced methyl ketones.

Actinobacteria are a large and diverse group of bacteria that are known to produce a wide range of secondary metabolites, many of which have important biological activities, including antibiotics, anti-cancer agents, and immunosuppressants. The biosynthesis of these compounds is often highly regulated with many natural products (NPs) being produced at very low levels in laboratory settings. Environmental factors, such as small molecule elicitors, can induce the production of secondary metabolites. Specifically, they can increase titers of known NPs as well as enabling discovery of novel NPs typically produced at undetectable levels. These elicitors can be NPs, including antibiotics or hormones, or synthetic compounds. In recent years, there has been a growing interest in the use of small molecule elicitors to induce the production of secondary metabolites from actinobacteria, especially for the discovery of NPs from "silent" biosynthetic gene clusters. This review aims to highlight classes of molecules that induce secondary metabolite production in actinobacteria and to describe the potential mechanisms of induction.

One-sentence summary: This review describes chemical elicitors of actinobacteria natural products described to date and the proposed mechanisms of induction.

Adaptive laboratory evolution (ALE) is a technique for the selection of strains with better phenotypes by long-term culture under a specific selection pressure or growth environment. Because ALE does not require detailed knowledge of a variety of complex and interactive metabolic networks, and only needs to simulate natural environmental conditions in the laboratory to design a selection pressure, it has the advantages of broad adaptability, strong practicability, and more convenient transformation of strains. In addition, ALE provides a powerful method for studying the evolutionary forces that change the phenotype, performance, and stability of strains, resulting in more productive industrial strains with beneficial mutations. In recent years, ALE has been widely used in the activation of specific microbial metabolic pathways and phenotypic optimization, the efficient utilization of specific substrates, the optimization of tolerance to toxic substance, and the biosynthesis of target products, which is more conducive to the production of industrial strains with excellent phenotypic characteristics. In this paper, typical examples of ALE applications in the development of industrial strains and the research progress of this technology are reviewed, followed by a discussion of its development prospects.

The successful design of economically viable bioprocesses can help to abate global dependence on petroleum, increase supply chain resilience, and add value to agriculture. Specifically, bioprocessing provides the opportunity to replace petrochemical production methods with biological methods and to develop novel bioproducts. Even though a vast range of chemicals can be biomanufactured, the constraints on economic viability, especially while competing with petrochemicals, are severe. There have been extensive gains in our ability to engineer microbes for improved production metrics and utilization of target carbon sources. The impact of growth medium composition on process cost and organism performance receives less attention in the literature than organism engineering efforts, with media optimization often being performed in proprietary settings. The widespread use of corn steep liquor as a nutrient source demonstrates the viability and importance of "waste" streams in biomanufacturing. There are other promising waste streams that can be used to increase the sustainability of biomanufacturing, such as the use of urea instead of fossil fuel-intensive ammonia and the use of struvite instead of contributing to the depletion of phosphate reserves. In this review, we discuss several process-specific optimizations of micronutrients that increased product titers by twofold or more. This practice of deliberate and thoughtful sourcing and adjustment of nutrients can substantially impact process metrics. Yet the mechanisms are rarely explored, making it difficult to generalize the results to other processes. In this review, we will discuss examples of nutrient sourcing and adjustment as a means of process improvement.

One-sentence summary: The potential impact of nutrient adjustments on bioprocess performance, economics, and waste valorization is undervalued and largely undercharacterized.

Coupling transcription of a cloned gene to the lac operon with induction by isopropylthio-β-galactoside (IPTG) has been a favoured approach for recombinant protein expression using Escherichia coli as a heterologous host for more than six decades. Despite a wealth of experimental data gleaned over this period, a quantitative relationship between extracellular IPTG concentration and consequent levels of recombinant protein expression remains surprisingly elusive across a broad spectrum of experimental conditions. This is because gene expression under lac operon regulation is tightly correlated with intracellular IPTG concentration due to allosteric regulation of the lac repressor protein (lacY). An in-silico mathematical model established that uptake of IPTG across the cytoplasmic membrane of E. coli by simple diffusion was negligible. Conversely, lacY mediated active transport was a rapid process, taking only some seconds for internal and external IPTG concentrations to equalize. Optimizing kcat and KM parameters by targeted mutation of the galactoside binding site in lacY could be a future strategy to improve the performance of recombinant protein expression. For example, if kcat were reduced whilst KM was increased, active transport of IPTG across the cytoplasmic membrane would be reduced, thereby lessening the metabolic burden on the cell and expediating accumulation of recombinant protein. The computational model described herein is made freely available and is amenable to optimize recombinant protein expression in other heterologous hosts.

One-sentence summary: A computational model made freely available to optimize recombinant protein expression in Escherichia coli other heterologous hosts.