Integrative transcriptome and proteome revealed high-yielding mechanisms of epsilon-poly-L-lysine by Streptomyces albulus.

Introduction: ε-poly-L-lysine (ε-PL) is a high value, widely used natural antimicrobial peptide additive for foods and cosmetic products that is mainly produced by Streptomyces albulus. In previous work, we developed the high-yield industrial strain S. albulus WG-608 through successive rounds of engineering. Methods: Here, we use integrated physiological, transcriptomic, and proteomics association analysis to resolve the complex mechanisms underlying high ε-PL production by comparing WG-608 with the progenitor strain M-Z18. Results: Our results show that key genes in the glycolysis, pentose phosphate pathway, glyoxylate pathway, oxidative phosphorylation, and L-lysine biosynthesis pathways are differentially upregulated in WG-608, while genes in the biosynthetic pathways for fatty acids, various branched amino acids, and secondary metabolite by-products are downregulated. This regulatory pattern results in the introduction of more carbon atoms into L-lysine biosynthesis and ε-PL production. In addition, significant changes in the regulation of DNA replication, transcription, and translation, two component systems, and quorum sensing may facilitate the adaptability to environmental pressure and the biosynthesis of ε-PL. Overexpression of ppk gene and addition of polyP6 further enhanced the ε-PL production. Discussion: This study enables comprehensive understanding of the biosynthetic mechanisms of ε-PL in S. albulus WG-608, while providing some genetic modification and fermentation strategies to further improve the ε-PL production.

Epsilon-poly-L-lysine: Recent Advances in Biomanufacturing and Applications.

ε-poly-L-lysine (ε-PL) is a naturally occurring poly(amino acid) of varying polymerization degree, which possesses excellent antimicrobial activity and has been widely used in food and pharmaceutical industries. To provide new perspectives from recent advances, this review compares several conventional and advanced strategies for the discovery of wild strains and development of high-producing strains, including isolation and culture-based traditional methods as well as genome mining and directed evolution. We also summarize process engineering approaches for improving production, including optimization of environmental conditions and utilization of industrial waste. Then, efficient downstream purification methods are described, including their drawbacks, followed by the brief introductions of proposed antimicrobial mechanisms of ε-PL and its recent applications. Finally, we discuss persistent challenges and future perspectives for the commercialization of ε-PL.

Extraction and purification of ε-poly-l-lysine from fermentation broth using an ethanol/ammonium sulfate aqueous two-phase system combined with ultrafiltration.

ε-Poly-l-lysine (ε-PL) serves as a natural food preservative and is manufactured mainly by extraction from microbial fermentation broth using ion-exchange chromatography. In order to develop an alternative purification strategy, an environmentally friendly alcohol/salt aqueous two-phase system (ATPS) was explored in this study for ε-PL extraction. A study of the separation of ε-PL in different alcohol/salt systems showed that ethanol/ammonium sulfate ATPS exhibited the highest ε-PL partition coefficient and recovery ratio. Based on the phase diagram, the effect of phase composition on partition, and the removal of pigment and protein, an ATPS that was composed of 20% (w/w) ethanol and 20% (w/w) ammonium sulfate, with a feedstock at pH 9.5, was developed to extract ε-PL from the fermentation broth. This achieved an ε-PL recovery ratio of 96.15% with an ε-PL purity of 40.23% after triplicate extractions. Subsequently, desalting by ultrafiltration led to a final ε-PL product of 92.39% purity and 87.72% recovery. The ethanol/ammonium sulfate ATPS provides a new possibility for ε-PL purification.

Mechanisms of response to pH shock in microbial fermentation.

The following review highlights pH shock, a novel environmental factor, as a tool for the improvement of fermentation production. The aim of this review is to introduce some recent original studies on the enhancement of microbial fermentation production by pH shock. Another purpose of this review is to improve the understanding of the processes that underlie physiological and genetic differences, which will facilitate future research on the improvement of fermentation production and reveal the associated molecular mechanisms. This understanding will simultaneously promote the application of this strategy to other microbial fermentation systems. Furthermore, improvement of the cellular tolerance of genetically engineered bacteria can also be a new field of research in the future to enhance fermentation production.

Full recycling of citric acid wastewater through anaerobic digestion, air-stripping and pH control.

Anaerobic digestion effluent (ADE) from the anaerobic digestion treatment of citric acid wastewater can be reused as a potential substitute for process water in the citric acid fermentation. However, excessive sodium contained in ADE significantly decreases citric acid production. In this paper, the inhibition mechanism of sodium on citric acid fermentation was investigated. We demonstrated that excessive sodium did not increase oxidative stress for Aspergillus niger, but reduced the pH of the medium significantly over the period 4-24 h, which led to lower activities of glucoamylase and isomaltase secreted by A. niger, with a decrease of available sugar concentration and citric acid production. ADE was pretreated by air-stripping prior to recycle and 18 g/L calcium carbonate was added at the start of fermentation to control the pH of the medium. The inhibition caused by ADE was completely alleviated and citric acid production substantially increased from 118.6 g/L to 141.4 g/L, comparable to the fermentation with deionized water (141.2 g/L). This novel process could decrease wastewater discharges and fresh water consumption in the citric acid industry, with benefit to the environment.

Understanding high ε-poly-L-lysine production by Streptomyces albulus using pH shock strategy in the level of transcriptomics.

ε-Poly-L-lysine (ε-PL) is a natural food preservative, which exhibits antimicrobial activity against a wide spectra of microorganisms. The production of ε-PL was significantly enhanced by pH shock in our previous study, but the underlying mechanism is poorly understood. According to transcriptional and physiological analyses in this study, the mprA/B and pepD signal transduction system was first proved to be presented and activated in Streptomyces albulus M-Z18 by pH shock, which positively regulated the transcription of ε-PL synthetase (Pls) gene and enhanced the Pls activity during fermentation. Furthermore, pH shock changed the ratio of unsaturation to saturation fatty acid in the membrane through up-regulating the transcription of fatty acid desaturase genes (SAZ_RS14940, SAZ_RS14945). In addition, pH shock also enhanced the transcription of cytochrome c oxidase (SAZ_RS15070, SAZ_RS15075), ferredoxin reductase (SAZ_RS34975) and iron sulfur protein (SAZ_RS31410) genes, and finally resulted in the improvement of cell respiratory activity. As a result, pH shock was considered to influence a wide range of proteins including regulators, fatty acid desaturase, respiratory chain component, and ATP-binding cassette transporter during fermentation. These combined influences might contribute to enhanced ε-PL productivity with pH shock.

Transcriptional study of the enhanced ε-poly-L-lysine productivity in culture using glucose and glycerol as a mixed carbon source.

A glucose-glycerol mixed carbon source (MCS) can substantially reduce batch fermentation time and improve ε-poly-L-lysine (ε-PL) productivity, which was of great significance in industrial microbial fermentation. This study aims to disclose the physiological mechanism by transcriptome analyses. In the MCS, the enhancements of gene transcription mainly emerged in central carbon metabolism, L-lysine synthesis as well as cell respiration, and these results were subsequently proved by quantitative real-time PCR assay. Intracellular L-lysine determination and exhaust gas analysis further confirmed the huge precursor L-lysine pool and active cell respiration in the MCS. Interestingly, in the MCS, pls was remarkably up-regulated than those in single carbon sources without transcriptional improvement of HrdD, which indicated that the improved ε-PL productivity was supported by other regulators rather than hrdD. This study exposed the physiological basis of the improved ε-PL productivity in the MCS, which provided references for studies on other biochemicals production using multiple substrates.

Effect of ion form of the ion-exchange resin on ε-poly-l-lysine purification from microbial fermentation broth.

ε-Poly-l-lysine (ε-PL) is an added-value natural product with widespread application in the fields of food, pharmaceuticals and biopolymer materials. However, the high production cost reduces its application. To improve the efficiency of ε-PL purification for decreasing the cost of downstream processes, the ion form of the ion-exchange resin, which is widely used for ε-PL purification, was investigated systematically in this study. Among eleven cation-exchange resins tested, the Amberlite IRC-50 resin offered the best adsorption capability and the highest desorption ratio. The adsorption kinetics of IRC-50 resin with H+, Na+ and NH4 + ion forms followed a pseudo-second-order model. The dynamic adsorption and desorption parameters of ε-PL were optimized with a column packed with IRC-50 resin with Na+ and NH4 +. It is suggested that NH4 + is the optimal ion form of IRC-50 resin for ε-PL extraction. Under optimal conditions, the IRC-50 resin with NH4 + achieved the highest ε-PL adsorption capability, purity and recovery ratio of 307.96 mg g-1, 76.52% and 96.2%, respectively. After further purification, a final ε-PL purity of 97.10% was achieved with a total recovery ratio of 66.01%. This is the first report on improving the ε-PL purification efficiency through optimizing the ion form of the ion-exchange resin. Moreover, it would offer guidance for other natural product recovery processes by ion-exchange chromatography.

Differential protein expression of a streptomycin-resistant Streptomyces albulus mutant in high yield production of ε-poly-l-lysine: a proteomics study.

ε-Poly-l-lysine (ε-PL), produced by Streptomyces albulus, is an excellent antimicrobial agent which has been extensively used in the field of food and medicine. In our previous study, we have improved ε-PL production by S. albulus M-Z18 through iterative introduction of streptomycin resistance. To decipher the overproduction mechanism of high-yielding mutant S. albulus SS-62, we conducted a comparative proteomics analysis between S. albulus SS-62 and its parent strain S. albulus M-Z18. Approximately 11.5% of the predicted S. albulus proteome was detected and 401 known or putative regulatory proteins showed statistically differential expression levels. Expression levels of proteins involved in ε-PL precursor metabolism and energy metabolism, and proteins in the pathways related to transcriptional regulation and translation were up-regulated. It was indicated that mutant SS-62 could not only strengthen the ε-PL precursor metabolism and energy metabolism but also tune the pathways related to transcriptional regulation and translation, suggesting a better intracellular metabolic environment for the synthesis of ε-PL in mutant SS-62. To confirm these bioinformatics analyses, qRT-PCR was employed to investigate the transcriptional levels of pls, frr and hrdD and their transcription levels were found to have increased more than 4-fold. Further, overexpression of pls and frr resulted in an increase in ε-PL titer and the yield of ε-PL per unit cell. This report not only represents the first comprehensive study on comparative proteomics in S. albulus, but it would also guide strain engineering to further improve ε-PL production.

Efficiently activated ε-poly-L-lysine production by multiple antibiotic-resistance mutations and acidic pH shock optimization in Streptomyces albulus.

ε-Poly-L-lysine (ε-PL) is a food additive produced by Streptomyces and is widely used in many countries. Working with Streptomyces albulus FEEL-1, we established a method to activate ε-PL synthesis by successive introduction of multiple antibiotic-resistance mutations. Sextuple mutant R6 was finally developed by screening for resistance to six antibiotics and produced 4.41 g/L of ε-PL in a shake flask, which is 2.75-fold higher than the level produced by the parent strain. In a previous study, we constructed a double-resistance mutant, SG-31, with high ε-PL production of 3.83 g/L and 59.50 g/L in a shake flask and 5-L bioreactor, respectively. However, we found that R6 did not show obvious advantages in fed-batch fermentation when compared with SG-31. For further activation of ε-PL synthesis ability, we optimized the fermentation process by using an effective acidic pH shock strategy, by which R6 synthetized 70.3 g/L of ε-PL, 2.79-fold and 1.18-fold greater than that synthetized by FEEL-1 and SG-31, respectively. To the best of our knowledge, this is the highest reported ε-PL production to date. This ε-PL overproduction may be due to the result of R99P and Q856H mutations in ribosomal protein S12 and RNA polymerase, respectively, which may be responsible for the increased transcription of the ε-poly-lysine synthetase gene (pls) and key enzyme activities in the Lys synthesis metabolic pathway. Consequently, ε-PL synthetase activity, intracellular ATP, and Lys concentrations were improved and directly contributed to ε-PL overproduction. This study combined ribosome engineering, high-throughput screening, and targeted strategy optimization to accelerate ε-PL production and probe the fermentation characteristics of hyperyield mutants. The information presented here may be useful for other natural products produced by Streptomyces.

Metabolic analyses of the improved ε-poly-L-lysine productivity using a glucose-glycerol mixed carbon source in chemostat cultures.

The glucose-glycerol mixed carbon source remarkably reduced the batch fermentation time of ε-poly-L-lysine (ε-PL) production, leading to higher productivity of both biomass and ε-PL, which was of great significance in industrial microbial fermentation. Our previous study confirmed the positive influence of fast cell growth on the ε-PL biosynthesis, while the direct influence of mixed carbon source on ε-PL production was still unknown. In this work, chemostat culture was employed to study the capacity of ε-PL biosynthesis in different carbon sources at a same dilution rate of 0.05 h-1. The results indicated that the mixed carbon source could enhance the ε-PL productivity besides the rapid cell growth. Analysis of key enzymes demonstrated that the activities of phosphoenolpyruvate carboxylase, citrate synthase, aspartokinase and ε-PL synthetase were all increased in chemostat culture with the mixed carbon source. In addition, the carbon fluxes were also improved in the mixed carbon source in terms of tricarboxylic acid cycle, anaplerotic and diaminopimelate pathway. Moreover, the mixed carbon source also accelerated the energy metabolism, leading to higher levels of energy charge and NADH/NAD+ ratio. The overall improvements of primary metabolism in chemostat culture with glucose-glycerol combination provided sufficient carbon skeletons and ATP for ε-PL biosynthesis. Therefore, the significantly higher ε-PL productivity in the mixed carbon source was a combined effect of both superior substrate group and rapid cell growth.

Insights into the simultaneous utilization of glucose and glycerol by Streptomyces albulus M-Z18 for high ε-poly-L-lysine productivity.

The simultaneous consumption of glucose and glycerol led to remarkably higher productivity of both biomass and ε-poly-L-lysine (ε-PL), which was of great significance in industrial microbial fermentation. To further understand the superior fermentation performances, transcriptional analysis and exogenous substrates addition were carried out to study the simultaneous utilization of glucose and glycerol by Streptomyces albulus M-Z18. Transcriptome analysis revealed that there was no mutual transcriptional suppression between the utilization of glucose and glycerol, which was quite different from typical "glucose effect". In addition, microorganisms cultivated with single glycerol showed significant demand for ribose-5-phosphate, which resulted in potential demand for glucose and xylitol. The above demand could be relieved by glucose (in the mixed carbon source) or xylitol addition, leading to improvement of biomass production. It indicated that glucose in the mixed carbon source was more important for biomass production. Besides, transcriptional analysis and exogenous citrate addition proved that single carbon sources could not afford enough carbon skeletons for Embden Meyerhof pathway (EMP) while a glucose-glycerol combination could provided sufficient carbon skeletons to saturate the metabolic capability of EMP, which contributed to the replenishment of precursors and energy consumed in ε-PL production. This study offered insight into the simultaneous consumption of glucose and glycerol in the ε-PL batch fermentation, which deepened our comprehension on the high ε-PL productivity in the mixed carbon source.

Development of Microtiter Plate Culture Method for Rapid Screening of ε-Poly-L-Lysine-Producing Strains.

ε-Poly-L-lysine (ε-PL) produced by Streptomyces albulus possesses a broad spectrum of antimicrobial activity and is widely used as a food preservative. To extensively screen ε-PL-overproducing strain, we developed an integrated high-throughput screening assay using ribosome engineering technology. The production protocol was scaled down to 24- and 48-deep-well microtiter plates (MTPs). The microplate reader assay was used to monitor ε-PL production. A good correlation was observed between the fermentation results obtained in both 24-(48)-deep-well MTPs and conventional Erlenmeyer flasks. Using this protocol, the production of ε-PL in an entire MTP was determined in <5 min without compromising on accuracy. The high-yielding strain selected through this protocol was also tested in Erlenmeyer flasks. The result showed that the ε-PL production of the high-yielding mutants was nearly 45% higher than that of the parent stain. Thus, development of this protocol is expected to accelerate the selection of ε-PL-overproducing strains.

Novel process combining anaerobic-aerobic digestion and ion exchange resin for full recycling of cassava stillage in ethanol fermentation.

A novel cleaner ethanol production process has been developed. Thin stillage is treated initially by anaerobic digestion followed by aerobic digestion and then further treated by chloride anion exchange resin. This allows the fully-digested and resin-treated stillage to be completely recycled for use as process water in the next ethanol fermentation batch, which eliminates wastewater discharges and minimizes consumption of fresh water. The method was evaluated at the laboratory scale. Process parameters were very similar to those found using tap water. Maximal ethanol production rate in the fully-recycled stillage was 0.9g/L/h, which was similar to the 0.9g/L/h found with the tap water control. The consumption of fresh water was reduced from 4.1L/L (fresh water/ethanol) to zero. Compared with anaerobically-aerobically digested stillage which had not been treated with resin, the fermentation time was reduced by 28% (from 72h to 52h) and reached the level achieved with tap water. This novel process can assist in sustainable development of the ethanol industry.

Enhanced ε-poly-L-lysine production by inducing double antibiotic-resistant mutations in Streptomyces albulus.

ε-Poly-L-lysine (ε-PL), as a food additive, has been widely used in many countries. However, its production still needs to be improved. We successfully enhanced ε-PL production of Streptomyces albulus FEEL-1 by introducing mutations related to antibiotics, such as streptomycin, gentamicin, and rifampin. Single- and double-resistant mutants (S-88 and SG-31) were finally screened with the improved ε-PL productions of 2.81 and 3.83 g/L, 1.75- to 2.39-fold compared with that of initial strain FEEL-1. Then, the performances of mutants S-88 and SG-31 were compared with the parent strain FEEL-1 in a 5-L bioreactor under the optimal condition for ε-PL production. After 174-h fed-batch fermentation, the ε-PL production and productivity of hyper-strain SG-31 reached the maximum of 59.50 g/L and 8.21 g/L/day, respectively, which was 138 and 105% higher than that of FEEL-1. Analysis of streptomycin-resistant mutants demonstrated that a point mutation occurred in rpsL gene (encoding the ribosomal protein S12). These single and double mutants displayed remarkable increases of the activities and transcriptional levels of key enzymes in ε-PL biosynthesis pathway, which may be responsible for the enhanced mycelia viability, respiratory activity, and ε-PL productions of SG-31. These results showed that the new breeding method, called ribosome engineering, could be a novel and effective breeding strategy for the evolution of ε-PL-producing strains.

Effect of acetic acid in recycling water on ethanol production for cassava in an integrated ethanol-methane fermentation process.

Recently, the integrated ethanol-methane fermentation process has been studied to prevent wastewater pollution. However, when the anaerobic digestion reaction runs poorly, acetic acid will accumulate in the recycling water. In this paper, we studied the effect of low concentration of acetic acid (≤25 mM) on ethanol fermentation at different initial pH values (4.2, 5.2 or 6.2). At an initial pH of 4.2, ethanol yields increased by 3.0% and glycerol yields decreased by 33.6% as the acetic acid concentration was increased from 0 to 25 mM. Raising the concentration of acetic acid to 25 mM increased the buffering capacity of the medium without obvious effects on biomass production in the cassava medium. Acetic acid was metabolized by Saccharomyces cerevisiae for the reason that the final concentration of acetic acid was 38.17% lower than initial concentration at pH 5.2 when 25 mM acetic acid was added. These results confirmed that a low concentration of acetic acid in the process stimulated ethanol fermentation. Thus, reducing the acetic acid concentration to a controlled low level is more advantageous than completely removing it.

Ethanol fermentation characteristics of recycled water by Saccharomyces cerevisiae in an integrated ethanol-methane fermentation process.

An process of integrated ethanol-methane fermentation with improved economics has been studied extensively in recent years, where the process water used for a subsequent fermentation of carbohydrate biomass is recycled. This paper presents a systematic study of the ethanol fermentation characteristics of recycled process water. Compared with tap water, fermentation time was shortened by 40% when mixed water was employed. However, while the maximal ethanol production rate increased from 1.07g/L/h to 2.01g/L/h, ethanol production was not enhanced. Cell number rose from 0.6×10(8) per mL in tap water to 1.6×10(8) per mL in mixed water but although biomass increased, cell morphology was not affected. Furthermore, the use of mixed water increased the glycerol yield but decreased that of acetic acid, and the final pH with mixed water was higher than when using tap water.

Genome Shuffling and Gentamicin-Resistance to Improve ε-Poly-L-Lysine Productivity of Streptomyces albulus W-156.

Genome shuffling has been a recently effective method for screening the desirable phenotypes of industrial strains. Here, we combined genome shuffling and gentamicin resistance to improve the production of ε-poly-L-lysine in Streptomyces albulus W-156. Five starting mutants with higher ε-poly-L-lysine (ε-PL) productivities were firstly obtained by atmospheric and room temperature plasma (ARTP) mutagenesis. After three rounds of genome shuffling with increasing concentration of gentamicin for selection, S. albulus AG3-28, was finally got with a production of 3.43 g/L in shaking flask. In a 5-L fermenter, AG3-28 exhibited a higher ε-PL productivity (56.5 g/L) than the initial strain W-156 (37.5 g/L). Key enzyme activities in primary and secondary metabolic pathways were analyzed, and the transcription levels of hrdD and pls were determined by quantitative real time-polymerase chain reaction (qRT-PCR). Increase of key enzyme activities and the upregulation of the gene transcriptional levels demonstrated that ε-PL synthetic pathway in AG3-28 was obviously strengthened, which might be responsible for the high productivity. Moreover, hyper-yield strain AG3-28 was found to produce a slightly lower ε-PL polymerization degree than the parent strain. Amplified fragment length polymorphism (AFLP) analysis reflects the genetic diversity among the derivates after genome shuffling.

A novel cleaner production process of citric acid by recycling its treated wastewater.

In this study, a novel cleaner production process of citric acid was proposed to completely solve the problem of wastewater management in citric acid industry. In the process, wastewater from citric acid fermentation was used to produce methane through anaerobic digestion and then the anaerobic digestion effluent was further treated with air stripping and electrodialysis before recycled as process water for the later citric acid fermentation. This proposed process was performed for 10 batches and the average citric acid production in recycling batches was 142.4±2.1g/L which was comparable to that with tap water (141.6g/L). Anaerobic digestion was also efficient and stable in operation. The average chemical oxygen demand (COD) removal rate was 95.1±1.2% and methane yield approached to 297.7±19.8mL/g TCODremoved. In conclusion, this novel process minimized the wastewater discharge and achieved the cleaner production in citric acid industry.

Effect of propionic acid on citric acid fermentation in an integrated citric acid-methane fermentation process.

In this study, an integrated citric acid-methane fermentation process was established to solve the problem of wastewater treatment in citric acid production. Citric acid wastewater was treated through anaerobic digestion and then the anaerobic digestion effluent (ADE) was further treated and recycled for the next batch citric acid fermentation. This process could eliminate wastewater discharge and reduce water resource consumption. Propionic acid was found in the ADE and its concentration continually increased in recycling. Effect of propionic acid on citric acid fermentation was investigated, and results indicated that influence of propionic acid on citric acid fermentation was contributed to the undissociated form. Citric acid fermentation was inhibited when the concentration of propionic acid was above 2, 4, and 6 mM in initial pH 4.0, 4.5 and, 5.0, respectively. However, low concentration of propionic acid could promote isomaltase activity which converted more isomaltose to available sugar, thereby increasing citric acid production. High concentration of propionic acid could influence the vitality of cell and prolong the lag phase, causing large amount of glucose still remaining in medium at the end of fermentation and decreasing citric acid production.