Enhanced Iturin A Production of Engineered Bacillus amyloliquefaciens by Knockout of Endogenous Plasmid and Rap Phosphatase Genes.

Iturin A biosynthesis has garnered considerable interest, yet bottlenecks persist in its low productivity in wild strains and the ability to engineer Bacillus amyloliquefaciens producers. This study reveals that deleting the endogenous plasmid, plas1, from the wild-type B. amyloliquefaciens HM618 notably enhances iturin A synthesis, likely related to the effect of the Rap phosphatase gene within plas1. Furthermore, inactivating Rap phosphatase-related genes (rapC, rapF, and rapH) in the genome of the strain also improved the iturin A level and specific productivity while reducing cell growth. Strategic rap genes and plasmid elimination achieved a synergistic balance between cell growth and iturin A production. Engineered strain HM-DR13 exhibited an increase in iturin A level to 849.9 mg/L within 48 h, significantly shortening the production period. These insights underscore the critical roles of endogenous plasmids and Rap phosphatases in iturin A biosynthesis, presenting a novel engineering strategy to optimize iturin A production in B. amyloliquefaciens.

Construction of yeast microbial consortia for petroleum hydrocarbons degradation.

Microbial degradation of petroleum hydrocarbons plays a vital role in mitigating petroleum contamination and heavy oil extraction. In this study, a Saccharomyces cerevisiae capable of degrading hexadecane has been successfully engineered, achieving a maximum degradation rate of up to 20.42%. However, the degradation ability of this strain decreased under various pressure conditions such as high temperature, high osmotic pressure, and acidity conditions. Therefore, a S. cerevisiae with high tolerance to these conditions has been constructed. And then, we constructed an "anti-stress hydrocarbon-degrading" consortium comprising engineered yeast strain SAH03, which degrades hexadecane, and glutathione synthetic yeast YGSH10, which provides stress resistance. This consortium was able to restore the degradation ability of SAH03 under various pressure conditions, particularly exhibiting a significant increase in degradation rate from 5.04% to 17.04% under high osmotic pressure. This study offers a novel approach for improving microbial degradation of petroleum hydrocarbons.

Biotransformation of ethylene glycol by engineered Escherichia coli.

There has been extensive research on the biological recycling of PET waste to address the issue of plastic waste pollution, with ethylene glycol (EG) being one of the main components recovered from this process. Therefore, finding ways to convert PET monomer EG into high-value products is crucial for effective PET waste recycling. In this study, we successfully engineered Escherichia coli to utilize EG and produce glycolic acid (GA), expecting to facilitate the biological recycling of PET waste. The engineered E. coli, able to utilize 10 g/L EG to produce 1.38 g/L GA within 96 h, was initially constructed. Subsequently, strategies based on overexpression of key enzymes and knock-out of the competing pathways are employed to enhance EG utilization along with GA biosynthesis. An engineered E. coli, characterized by the highest GA production titer and substrate conversion rate, was obtained. The GA titer increased to 5.1 g/L with a yield of 0.75 g/g EG, which is the highest level in the shake flake experiments. Transcriptional level analysis and metabolomic analysis were then conducted, revealing that overexpression of key enzymes and knock-out of the competing pathways improved the metabolic flow in the EG utilization. The improved metabolic flow also leads to accelerated synthesis and metabolism of amino acids.

Integrated Biofilm Modification and Transcriptional Analysis for Improving Fengycin Production in Bacillus amyloliquefaciens.

Although fengycin exhibits broad-spectrum antifungal properties, its application is hindered due to its low biosynthesis level and the co-existence of iturin A and surfactin in Bacillus amyloliquefaciens HM618, a probiotic strain. In this study, transcriptome analysis and gene editing were used to explore the potential mechanisms regulating fengycin production in B. amyloliquefaciens. The fengycin level of B. amyloliquefacien HM-3 (∆itu-ΔsrfAA) was 88.41 mg/L after simultaneously inhibiting the biosyntheses of iturin A and surfactin. The knockout of gene eps associated with biofilm formation significantly increased the fengycin level of the strain HM618, whereas the fengycin level decreased 32.05% after knocking out sinI, a regulator of biofilm formation. Transcriptome analysis revealed that the differentially expressed genes, involved in pathways of amino acid and fatty acid syntheses, were significantly down-regulated in the recombinant strains, which is likely associated with a decrease of fengycin production. The knockout of gene comQXPA and subsequent transcriptome analysis revealed that the ComQXPA quorum sensing system played a positive regulatory role in fengycin production. Through targeted genetic modifications and fermentation optimization, the fengycin production of the engineered strain HM-12 (∆itu-ΔsrfAA-ΔyvbJ) in a 5-L fermenter reached 1.172 g/L, a 12.26-fold increase compared to the fengycin level in the strain HM-3 (∆itu-ΔsrfAA) in the Erlenmeyer flask. Taken together, these results reveal the underlying metabolic mechanisms associated with fengycin synthesis and provide a potential strategy for improving fengycin production in B. amyloliquefaciens.

Improving salt-tolerant artificial consortium of Bacillus amyloliquefaciens for bioconverting food waste to lipopeptides.

High-salt content in food waste (FW) affects its resource utilization during biotransformation. In this study, adaptive laboratory evolution (ALE), gene editing, and artificial consortia were performed out to improve the salt-tolerance of Bacillus amyloliquefaciens for producing lipopeptide under FW and seawater. High-salt stress significantly decreased lipopeptide production in the B. amyloliquefaciens HM618 and ALE strains. The total lipopeptide production in the recombinant B. amyloliquefaciens HM-4KSMSO after overexpressing the ion transportor gene ktrA and proline transporter gene opuE and replacing the promoter of gene mrp was 1.34 times higher than that in the strain HM618 in medium containing 30 g/L NaCl. Lipopeptide production under salt-tolerant consortia containing two strains (HM-4KSMSO and Corynebacterium glutamicum) and three-strains (HM-4KSMSO, salt-tolerant C. glutamicum, and Yarrowia lipolytica) was 1.81- and 2.28-fold higher than that under pure culture in a medium containing FW or both FW and seawater, respectively. These findings provide a new strategy for using high-salt FW and seawater to produce value-added chemicals.

An Engineered Microbial Consortium Provides Precursors for Fengycin Production by Bacillus subtilis.

Fengycin has great potential for applications in biological control because of its biosafety and degradability. In this study, the addition of exogenous precursors increased fengycin production by Bacillus subtilis. Corynebacterium glutamicum was engineered to produce high levels of precursors (Thr, Pro, Val, and Ile) to promote the biosynthesis of fengycin. Furthermore, recombinant C. glutamicum and Yarrowia lipolytica providing amino acid and fatty acid precursors were co-cultured to improve fengycin production by B. subtilis in a three-strain artificial consortium, in which fengycin production was 2100 mg·L-1. In addition, fengycin production by the consortium in a 5 L bioreactor reached 3290 mg·L-1. Fengycin had a significant antifungal effect on Rhizoctonia solani, which illustrates its potential as a food preservative. Taken together, this work provides a new strategy for improving fengycin production by a microbial consortium and metabolic engineering.

Enhancing fengycin production in the co-culture of Bacillus subtilis and Corynebacterium glutamicum by engineering proline transporter.

Fengycin possesses antifungal activity but has limited application due to its low yields. Amino acid precursors play a crucial role in fengycin synthesis. Herein, the overexpression of alanine, isoleucine, and threonine transporter-related genes in Bacillus subtilis increased fengycin production by 34.06%, 46.66%, and 7.83%, respectively. Particularly, fengycin production in B. subtilis reached 871.86 mg/L with the addition of 8.0 g/L exogenous proline after enhancing the expression of the proline transport-related gene opuE. To overcome the metabolic burden caused by excessive enhancement of gene expression for supplying precursors, B. subtilis and Corynebacterium glutamicum which produced proline, were co-cultured, which further improved fengycin production. Fengycin production in the co-culture of B. subtilis and C. glutamicum in shake flasks reached 1554.74 mg/L after optimizing the inoculation time and ratio. The fengycin level in the fed-batch co-culture was 2309.96 mg/L in a 5.0-L bioreactor. These findings provide a new strategy for improving fengycin production.

Design and construction of microbial cell factories based on systems biology.

Environmental sustainability is an increasingly important issue in industry. As an environmentally friendly and sustainable way, constructing microbial cell factories to produce all kinds of valuable products has attracted more and more attention. In the process of constructing microbial cell factories, systems biology plays a crucial role. This review summarizes the recent applications of systems biology in the design and construction of microbial cell factories from four perspectives, including functional genes/enzymes discovery, bottleneck pathways identification, strains tolerance improvement and design and construction of synthetic microbial consortia. Systems biology tools can be employed to identify functional genes/enzymes involved in the biosynthetic pathways of products. These discovered genes are introduced into appropriate chassis strains to build engineering microorganisms capable of producing products. Subsequently, systems biology tools are used to identify bottleneck pathways, improve strains tolerance and guide design and construction of synthetic microbial consortia, resulting in increasing the yield of engineered strains and constructing microbial cell factories successfully.

Bioengineering for the Microbial Degradation of Petroleum Hydrocarbon Contaminants.

Petroleum hydrocarbons are relatively recalcitrant compounds, and as contaminants, they are one of the most serious environmental problems. n-Alkanes are important constituents of petroleum hydrocarbons. Advances in synthetic biology and metabolic engineering strategies have made n-alkane biodegradation more designable and maneuverable for solving environmental pollution problems. In the microbial degradation of n-alkanes, more and more degradation pathways, related genes, microbes, and alkane hydroxylases have been discovered, which provide a theoretical basis for the further construction of degrading strains and microbial communities. In this review, the current advances in the microbial degradation of n-alkanes under aerobic condition are summarized in four aspects, including the biodegradation pathways and related genes, alkane hydroxylases, engineered microbial chassis, and microbial community. Especially, the microbial communities of "Alkane-degrader and Alkane-degrader" and "Alkane-degrader and Helper" provide new ideas for the degradation of petroleum hydrocarbons. Surfactant producers and nitrogen providers as a "Helper" are discussed in depth. This review will be helpful to further achieve bioremediation of oil-polluted environments rapidly.

Construction of microbial consortia for microbial degradation of complex compounds.

Increasingly complex synthetic environmental pollutants are prompting further research into bioremediation, which is one of the most economical and safest means of environmental restoration. From the current research, using microbial consortia to degrade complex compounds is more advantageous compared to using isolated bacteria, as the former is more adaptable and stable within the growth environment and can provide a suitable catalytic environment for each enzyme required by the biodegradation pathway. With the development of synthetic biology and gene-editing tools, artificial microbial consortia systems can be designed to be more efficient, stable, and robust, and they can be used to produce high-value-added products with their strong degradation ability. Furthermore, microbial consortia systems are shown to be promising in the degradation of complex compounds. In this review, the strategies for constructing stable and robust microbial consortia are discussed. The current advances in the degradation of complex compounds by microbial consortia are also classified and detailed, including plastics, petroleum, antibiotics, azo dyes, and some pollutants present in sewage. Thus, this paper aims to support some helps to those who focus on the degradation of complex compounds by microbial consortia.

Improved Production of Fengycin in Bacillus subtilis by Integrated Strain Engineering Strategy.

Fengycin is a lipopeptide with broad-spectrum antifungal activity. However, its low yield limits its commercial application. Therefore, we iteratively edited multiple target genes associated with fengycin synthesis by combinatorial metabolic engineering. The ability of Bacillus subtilis 168 to manufacture lipopeptides was restored, and the fengycin titer was 1.81 mg/L. Fengycin production was further increased to 174.63 mg/L after knocking out pathways associated with surfactin and bacillaene synthesis and replacing the native promoter (PppsA) with the Pveg promoter. Subsequently, fengycin levels were elevated to 258.52 mg/L by upregulating the expression of relevant genes involved in the fatty acid pathway. After blocking spore and biofilm formation, fengycin production reached 302.51 mg/L. Finally, fengycin production was increased to approximately 885.37 mg/L after adding threonine in the optimized culture medium, which was 488-fold higher compared with that of the initial strain. Integrated strain engineering provides a strategy to construct a system for improving fengycin production.

Current Advances in Biodegradation of Polyolefins.

Polyolefins, including polyethylene (PE), polypropylene (PP) and polystyrene (PS), are widely used plastics in our daily life. The excessive use of plastics and improper handling methods cause considerable pollution in the environment, as well as waste of energy. The biodegradation of polyolefins seems to be an environmentally friendly and low-energy consumption method for plastics degradation. Many strains that could degrade polyolefins have been isolated from the environment. Some enzymes have also been identified with the function of polyolefin degradation. With the development of synthetic biology and metabolic engineering strategies, engineered strains could be used to degrade plastics. This review summarizes the current advances in polyolefin degradation, including isolated and engineered strains, enzymes and related pathways. Furthermore, a novel strategy for polyolefin degradation by artificial microbial consortia is proposed, which would be helpful for the efficient degradation of polyolefin.

Construction of synthetic microbial consortia for 2-keto-L-gulonic acid biosynthesis.

Currently, the establishment of synthetic microbial consortia with rational strategies has gained extensive attention, becoming one of the important frontiers of synthetic biology. Systems biology can offer insights into the design and construction of synthetic microbial consortia. Taking the high-efficiency production of 2-keto-l-gulonic acid (2-KLG) as an example, we constructed a synthetic microbial consortium "Saccharomyces cerevisiae-Ketogulonigenium vulgare" based on systems biology analysis. In the consortium, K. vulgare was the 2-KLG producing strain, and S. cerevisiae acted as the helper strain. Comparative transcriptomic analysis was performed on an engineered S. cerevisiae (VTC2) and a wild-type S. cerevisiae BY4741. The results showed that the up-regulated genes in VTC2, compared with BY4741, were mainly involved in glycolysis, TCA cycle, purine metabolism, and biosynthesis of amino acids, B vitamins, and antioxidant proteases, all of which play important roles in promoting the growth of K. vulgare. Furthermore, Vitamin C produced by VTC2 could further relieve the oxidative stress in the environment to increase the production of 2-KLG. Therefore, VTC2 would be of great advantage in working with K. vulgare. Thus, the synthetic microbial consortium "VTC2-K. vulgare" was constructed based on transcriptomics analyses, and the accumulation of 2-KLG was increased by 1.49-fold compared with that of mono-cultured K. vulgare, reaching 13.2 ± 0.52 g/L. In addition, the increased production of 2-KLG was accompanied by the up-regulated activities of superoxide dismutase and catalase in the medium and the up-regulated oxidative stress-related genes (sod, cat and gpd) in K. vulgare. The results indicated that the oxidative stress in the synthetic microbial consortium was efficiently reduced. Thus, systems analysis confirmed a favorable symbiotic relationship between microorganisms, providing guidance for further engineering synthetic consortia.

Androgen receptor transactivates KSHV noncoding RNA PAN to promote lytic replication-mediated oncogenesis: A mechanism of sex disparity in KS.

Kaposi's sarcoma-associated herpesvirus (KSHV) preferentially infects and causes Kaposi's sarcoma (KS) in male patients. However, the biological mechanisms are largely unknown. This study was novel in confirming the extensive nuclear distribution of the androgen receptor (AR) and its co-localization with viral oncoprotein of latency-associated nuclear antigen in KS lesions, indicating a transcription way of AR in KS pathogenesis. The endogenous AR was also remarkably higher in KSHV-positive B cells than in KSHV-negative cells and responded to the ligand treatment of 5α-dihydrotestosterone (DHT), the agonist of AR. Then, the anti-AR antibody-based chromatin immunoprecipitation (ChIP)-associated sequencing was used to identify the target viral genes of AR, revealing that the AR bound to multiple regions of lytic genes in the KSHV genome. The highest peak was enriched in the core promoter sequence of polyadenylated nuclear RNA (PAN), and the physical interaction was verified by ChIP-polymerase chain reaction (PCR) and the electrophoretic mobility shift assay (EMSA). Consistently, male steroid treatment significantly transactivated the promoter activity of PAN in luciferase reporter assay, consequently leading to extensive lytic gene expression and KSHV production as determined by real-time quantitative PCR, and the deletion of nuclear localization signals of AR resulted in the loss of nuclear transport and transcriptional activity in the presence of androgen and thus impaired the expression of PAN RNA. Oncogenically, this study identified that the AR was a functional prerequisite for cell invasion, especially under the context of KSHV reactivation, through hijacking the PAN as a critical effector. Taken together, a novel mechanism from male sex steroids to viral noncoding RNA was identified, which might provide a clue to understanding the male propensity in KS.

[Advances in biodegradation of petroleum hydrocarbons].

Petroleum hydrocarbon pollutants are difficult to be degraded, and bioremediation has received increasing attention for remediating the hydrocarbon polluted area. This review started by introducing the interphase adaptation and transport process of hydrocarbon by microbes. Subsequently, the advances made in the identification of hydrocarbon-degrading strains and genes as well as elucidation of metabolic pathways and underpinning mechanisms in the biodegradation of typical petroleum hydrocarbon pollutants were summarized. The capability of wild-type hydrocarbon degrading bacteria can be enhanced through genetic engineering and metabolic engineering. With the rapid development of synthetic biology, the bioremediation of hydrocarbon polluted area can be further improved by engineering the metabolic pathways of hydrocarbon-degrading microbes, or through design and construction of synthetic microbial consortia.

One-Step Biosynthesis of Vitamin C in Saccharomyces cerevisiae.

Vitamin C (VC) is comprehensively applied in foods, cosmetics, pharmaceuticals, and especially clinical medicine. Nowadays, the industrial production of VC mainly relies on the classic two-step fermentation route, and researchers have explored the way for one-step fermentation of VC in recent years. In this study, a VC biosynthesis pathway that directly produced VC from glucose was reconstructed in Saccharomyces cerevisiae, and the protein engineering and metabolic engineering strategies were adopted to improve it. First, five exogenous modules from Arabidopsis were introduced into the chassis cells by synthetic biology approaches to obtain the strain YLAA harboring VC biosynthesis. In addition, L-galactose dehydrogenase (L-GalDH) and L-galactono-1,4-lactone dehydrogenase (L-GLDH) were fused and expressed in S. cerevisiae cells for the first time, which increased the intracellular VC accumulation by 2.78-fold, reaching 9.97 ± 0.09 mg/L. Through copy number engineering, it was further confirmed that the last step catalyzed by L-GLDH is the rate-limiting step. GDP-L-galactose phosphorylase (GPP) encoded by vtc2 is another rate-limiting enzyme confirmed by GAL1p overexpression results. Finally, by balancing gene expression and cell growth, the highest production strain with overexpressing vtc2 by multicopy plasmids was constructed. The VC accumulation reached 24.94 ± 1.16 mg/L, which was currently the highest production from glucose in S. cerevisiae. The production of the recombinant strain reached nearly 44 mg/L with the exogenous addition of L-galactose or glutathione. The results further emphasized the importance of the step catalyzed by GPP. The investigation provided experience for the efficient biosynthesis of VC and the determination of rate-limiting steps.

Evaluation of PET Degradation Using Artificial Microbial Consortia.

Polyethylene terephthalate (PET) biodegradation is regarded as an environmentally friendly degradation method. In this study, an artificial microbial consortium composed of Rhodococcus jostii, Pseudomonas putida and two metabolically engineered Bacillus subtilis was constructed to degrade PET. First, a two-species microbial consortium was constructed with two engineered B. subtilis that could secrete PET hydrolase (PETase) and monohydroxyethyl terephthalate hydrolase (MHETase), respectively; it could degrade 13.6% (weight loss) of the PET film within 7 days. A three-species microbial consortium was further obtained by adding R. jostii to reduce the inhibition caused by terephthalic acid (TPA), a breakdown product of PET. The weight of PET film was reduced by 31.2% within 3 days, achieving about 17.6% improvement compared with the two-species microbial consortium. Finally, P. putida was introduced to reduce the inhibition caused by ethylene glycol (EG), another breakdown product of PET, obtaining a four-species microbial consortium. With the four-species consortium, the weight loss of PET film reached 23.2% under ambient temperature. This study constructed and evaluated the artificial microbial consortia in PET degradation, which demonstrated the great potential of artificial microbial consortia in the utilization of complex substrates, providing new insights for biodegradation of complex polymers.

Current Advances in the Biodegradation and Bioconversion of Polyethylene Terephthalate.

Polyethylene terephthalate (PET) is a widely used plastic that is polymerized by terephthalic acid (TPA) and ethylene glycol (EG). In recent years, PET biodegradation and bioconversion have become important in solving environmental plastic pollution. More and more PET hydrolases have been discovered and modified, which mainly act on and degrade the ester bond of PET. The monomers, TPA and EG, can be further utilized by microorganisms, entering the tricarboxylic acid cycle (TCA cycle) or being converted into high value chemicals, and finally realizing the biodegradation and bioconversion of PET. Based on synthetic biology and metabolic engineering strategies, this review summarizes the current advances in the modified PET hydrolases, engineered microbial chassis in degrading PET, bioconversion pathways of PET monomers, and artificial microbial consortia in PET biodegradation and bioconversion. Artificial microbial consortium provides novel ideas for the biodegradation and bioconversion of PET or other complex polymers. It is helpful to realize the one-step bioconversion of PET into high value chemicals.

Crocetin Overproduction in Engineered Saccharomyces cerevisiae via Tuning Key Enzymes Coupled With Precursor Engineering.

Crocetin, an important natural carotenoid dicarboxylic acid with high pharmaceutical values, has been successfully generated from glucose by engineered Saccharomyces cerevisiae in our previous study. Here, a systematic optimization was executed for crocetin overproduction in yeast. The effects of precursor enhancement on crocetin production were investigated by blocking the genes involved in glyoxylate cycle [citric acid synthase (CIT2) and malic acid synthase (MLS1)]. Crocetin titer was promoted by 50% by ΔCIT2 compared to that of the starting strain. Then, the crocetin production was further increased by 44% through introducing the forward fusion enzymes of PsCrtZ (CrtZ from Pantoea stewartii)-CsCCD2 (CCD2 from Crocus sativus). Consequently, the crocetin titer reached to 1.95 ± 0.23 mg/L by overexpression of PsCrtZ-CsCCD2 followed by medium optimization. Eventually, a titer of 12.43 ± 0.62 mg/L crocetin was achieved in 5-L bioreactor, which is the highest crocetin titer reported in micro-organisms.

Exploring Catalysis Specificity of Phytoene Dehydrogenase CrtI in Carotenoid Synthesis.

Carotenoids, a variety of natural products, have significant pharmaceutical and commercial potential. Phytoene dehydrogenase (CrtI) is the rate-limit enzyme for carotenoid synthesis, whose catalysis specificity results in various carotenoids. However, the structural characteristics of CrtI for controlling the catalysis specificity on dehydrogenation steps are still unclear, which limited the development of CrtI function. Here we confirmed two mutation sites H136 and H453 in the mutant library of CrtI from Blakeslea trispora, which markedly regulated catalytic specificity. Interestingly, the sequence alignment features at H136 and H453 were consistent with the phylogenetic analysis of CrtI families. Subsequently, the functions of saturated mutants at H136 and H453 were clustered by principal component analysis (PCA) and k-means. According to the clustering results, diversiform mutants with specific dehydrogenation function provided important application value for carotenoid product customization. Meanwhile, this study also enriched the theory of enzyme evolution and guided the functional development of enzymes.