Enhanced production of ε-poly-L-lysine by immobilized Streptomyces ahygroscopicus through repeated-batch or fed-batch fermentation with in situ product removal.

ε-Poly-L-lysine (ε-PL) is a naturally-occurring L-lysine homopolymer having a broad-spectrum antimicrobial activity and used widely as a food preservative. In the present study, the combined use of immobilization and in situ product removal (ISPR) was evaluated for the production of ε-PL by Streptomyces ahygroscopicus GIM8. Results showed that ε-PL production in the flask cultures decreased from 0.84 to 0.38-0.56 g/L upon immobilization on loofah sponge with different amounts (0.5-3 g in 50 mL medium in a flask). By applying continuous ISPR to the immobilized flask cultures, ε-PL production as high as 3.51 g/L was obtained compared to 0.51 g/L of the control. A satisfactory titer of 1.84 g/L ε-PL could also be achieved with intermittent ISRP (three cycles of ISPR operation during cultivation). Further investigation showed that low levels of ε-PL retained in the broth appeared to favor its biosynthesis. In the repeated-batch fermentation in a 5 L immobilized bioreactor, with continuous ISPR, the final average ε-PL concentration and productivity were 3.35 g/L and 0.797 g/L/day, respectively, and 3.18 g/L and 0.756 g/L/day for the alternative (intermittent ISPR), in comparison to 1.16 g/L and 0.277 g/L/day with no ISPR usage. In the fed-batch fermentation with immobilized cells, the combined use of intermittent ISPR and extra nutrient feeding increased ε-PL concentration and productivity up to 24.57 g/L and 9.34 g/L/day. The fermentation processes developed could serve as an effective approach for ε-PL production and, moreover, the combination could greatly simplify downstream processing for ε-PL separation and purification.

Enhancement of ε-poly-L-lysine production in Streptomyces griseofuscus by addition of exogenous astaxanthin.

Addition of exogenous astaxanthin for improving ε-poly-L-lysine (ε-PL) production in Streptomyces griseofuscus was investigated in this study. By this unique strategy, the ε-PL production in shaker-flask fermentation was 2.48 g/L, which was 67.5% higher than the control at the addition dosage of 1.0 g/L, owing to the oxidation resistance of astaxanthin. In fed-batch fermentation, the ε-PL production reached 36.1 g/L, a 36.3% increase compared to the control. Intracellular response for oxidation in S. griseofuscus such as ROS generation and lipid peroxidation was reduced by astaxanthin addition. Illumina RNA deep sequencing (RNA-seq) technology further revealed that S. griseofuscus with astaxanthin addition showed down-regulated transcriptions of genes involved in oxidative stress. This research proved that the beneficial effect of astaxanthin addition was far better than glutathione (GSH) owing to the stronger antioxidant capacity, and provided a novel approach to regulate ε-PL synthesis.

Enhancement of metabolic flux toward ε-poly-l-lysine biosynthesis by targeted inactivation of concomitant polyene macrolide biosynthesis in Streptomyces albulus.

ε-Poly-l-lysine (ε-PL) produced as a secondary metabolite of Streptomyces albulus has long been used as a natural food preservative in a number of countries, including Japan, the United States, South Korea, and China. To date, numerous studies employing classical biotechnological approaches have been carried out to improve its productivity. Here we report a modern and rational genetic approach to enhancing metabolic flux toward ε-PL biosynthesis. Based on in silico genome analyses, we revealed that S. albulus NBRC14147 produces five antifungal polyene antibiotics-tetramycin A and B, tetrin A and B, and a trace amount of nystatin A1-concomitantly with antimicrobial ε-PL. Targeted inactivation of the biosynthetic gene cluster for tetramycins and tetrins in a nystatin A1 production-deficient mutant completely abolished the production of polyene macrolides, which in turn led to an approximately 20% improvement in ε-PL production that closely correlated with the polyene defects. The biosynthetic flux for ε-PL was thus successfully enhanced by inactivation of the concomitant secondary metabolite biosynthetic pathways. Since this elimination of concomitantly produced metabolites also allows for simpler purification after fermentation production of ε-PL, the rational strain engineering strategy we show here will improve its industrial production.

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.

The potential of a natural biopolymeric flocculant, ε-poly-L-lysine, for harvesting Chlorella ellipsoidea and its sustainability perspectives for cost and toxicity.

The successful production of microalgal biomass requires the precise coordination of many different steps. Cell harvesting is a central process in all methods currently used for the production of microalgal biomass. Therefore, improving the harvesting process itself, and using a harvesting method that is compatible with adjacent steps, is necessary to prevent problems that may occur during downstream processing. This study examined the potential of the cationic biopolymer ε-poly-L-lysine (ε-PLL) for use in the harvest of microalgae (Chlorella ellipsoidea). The effects of ε-PLL concentration and mixing intensity on flocculation efficiency and operating costs were determined. We found that ε-PLL was not toxic to microalgal cells at concentrations of up to 25 mg/L, based on the photosystem II quantum yield. A recovery rate of 95% was achieved using 19 mg/L ε-PLL, and the estimated harvest cost was 20 US$/ton of harvested biomass. Moreover, ε-PLL displayed antimicrobial properties, leaving the harvested biomass intact and pure. Therefore, the use of ε-PLL-induced flocculation appears to be an attractive option when harvesting microalgal biomass for use as low- and high-value commodities for humans or animals.

Discovery of a Short-Chain ε-Poly-l-lysine and Its Highly Efficient Production via Synthetase Swap Strategy.

ε-Poly-l-lysine (ε-PL) is a natural antimicrobial cationic peptide, which is generally recognized as safe for use as a food preservative. To date, the production capacity of strains that produce low-molecular weight ε-PL remains very low and thus unsuitable for industrial production. Here, we report a new low-molecular weight ε-PL-producing Kitasatospora aureofaciens strain. The ε-PL synthase gene of this strain was cloned into a high ε-PL-producing Streptomyces albulus strain. The resulting recombinant strain efficiently produced ε-PL with a molecular weight of 1.3-2.3 kDa and yielded of 23.6 g/L following fed-batch fermentation in a 5 L bioreactor. In addition, circular dichroism spectra showed that this ε-PL takes on a conformation similar to an antiparallel pleated-sheet. Moreover, it demonstrated better antimicrobial activity against yeast compared to the 3.2-4.5 kDa ε-PL. This study provides a highly efficient strategy for production of the low-molecular weight ε-PL, which helps to expand its potential applications.

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.

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.

Chlamydomonas sp. as dynamic biorefinery feedstock for the production of methyl ester and ɛ-polylysine.

An integrated production of methyl ester and ɛ-polylysine from Chlamydomonas sp. was studied using biorefinery approach. The harvesting efficiency of Chlamydomonas sp. was increased up to 92% by treatment with a flocculant FeCl3 at 100 mg/L for 30 min. The DMC (dimethyl carbonate) mediated enzyme catalyzed in-situ transesterification of Chlamydomonas sp. yielded the maximum methyl ester of 92% under optimized conditions. The valued-added product ɛ-polylysine was produced from hydrolysate obtained from the spent biomass of Chlamydomonas sp. using Streptomyces sp. The key components of sugar and MgSO4 used for ɛ-polysine production were optimized whereby the maximum ɛ-polylysine production was achieved at 50 g/L sugar and 0.3 g/L MgSO4. The ɛ-polylysine production was further enhanced by supplementation of important amino acids (lysine and aspartate) and TCA cycle intermediates (citric acid and α-ketoglutaric acid). The maximum ɛ-polylysine production of 2.24 g/L was found with 4 mM citric acid supplementation after 110 h.

Streptomyces albulus yields ε-poly-L-lysine and other products from salt-contaminated glycerol waste.

Actinomycetes are the most important microorganisms for the industrial production of secondary metabolites with antimicrobial and anticancer properties. However, they have not been implicated in biorefineries. Here, we study the ability of the ε-poly-L-lysine producing Streptomyces albulus BCRC 11814 to utilize biodiesel-derived crude glycerol. S. albulus was cultured in a mineral medium supplemented with up to 10% w/v sodium chloride or potassium chloride, and with crude glycerol as the sole carbohydrate source. Under these conditions, the strain produced 0.1 g ε-poly-L-lysine per 1 g of biomass. RNA sequencing revealed upregulation of the ectoine biosynthetic pathway of S. albulus, which provides proof of halotolerance. S. albulus has several silent secondary metabolite biosynthetic clusters predicted within the genome. Based on the results, we conclude that S. albulus BCRC 11814 is a halotolerant microorganism capable of utilizing biodiesel-derived crude glycerol better than other actinomycetes included in the present study. S. albulus has the potential to be established as microbial platform production host for a range of high-value biological products.

Enhancement of ε-poly-L-lysine production by overexpressing the ammonium transporter gene in Streptomyces albulus PD-1.

The antibacterial polymer ɛ-poly-L-lysine (ε-PL) has been widely used as a safe food preservative. As the synthesis of ε-PL requires a rich supply of nitrogen, the efficiency of nitrogen translocation and utilization is extremely important. The objective of this study was to improve the production of ε-PL by overexpressing the ammonium transporter gene amtB in Streptomyces albulus PD-1. Using the recombinant bacteria, the optimum carbon-to-nitrogen ratio in the synthesis stage of fermentation increased from 3 to 4.71, compared with that obtained using the wild-type strain, and the utilization efficiency of ammonium was improved too. Ultimately, the production of ε-PL increased from 22.7 to 35.7 g/L upon fed-batch cultivation in a 5 L bioreactor. Determination of the expression of the genes and enzymes associated with ammonium metabolism and ε-PL synthesis revealed that the overexpression of amtB in S. albulus PD-1 enhanced ε-PL biosynthesis by increasing the activity of the corresponding metabolic pathways. To the best of our knowledge, this is the first report on enhancing ε-PL production by overexpression of the amtB gene in an ε-PL-producing strain.

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.

Medium optimization for ε-poly-L-lysine production by Streptomyces diastatochromogenes using response surface methodology.

Poly-ε-L-lysine is a natural homo-polyamide of L-lysine with excellent antimicrobial properties, which can be used as a novel preservative and has a wide range of applications. In this paper, the fermentation medium for ε-PL production by Streptomyces diastatochromogenes 6#-7 was optimized by Response Surface Methodology. The results of Plackett-Burman design showed that glucose, yeast extract and (NH4 )2 SO4 were the major influencing factors in ε-PL production of S. diastatochromogenes 6#-7. The optimal concentrations of glucose, yeast extract and (NH4 )2 SO4 were determined to be 60, 7·5 and 7·5 g l-1 according to Box-Behnken experiment and regression analysis, respectively. Under the optimized conditions, the ε-PL yield in shake-flask fermentation was 0·948 ± 0·030 g l-1 , which was in good agreement with the predicted value of 0·970 g l-1 . The yield was improved by 43·1% from that with the initial medium. In 5 l jar-fermenter the ε-PL yield reached 25·5 g l-1 , which was increased by 56·4% from the original medium. In addition, the fermentation time was reduced from 174 to 120 h. SIGNIFICANCE AND IMPACT OF THE STUDY: Medium optimization is a very practical and valuable tool for fermentation industry to improve product yield and minimize by-products as well as reduce overall manufacturing costs. The response surface methodology is not new, but it is still a very effective method in medium optimization research. This study used ε-polylysine fermentation as an example to demonstrate how the product yield can be significantly increased by medium optimization through surface response methodology. Similar approach can be used in other microbial fermentations such as in pharmaceutical, food, agricultural and energy industries. As an example, ε-polylysine is one of a few newly approved natural food-grade antimicrobials for food and beverages preservations. Yield improvement is economically beneficial to not only ε-polylysine manufacturers but also to their users and consumers due to lower costs and price.

Enhancement of ε-poly-L-lysine synthesis in Streptomyces by exogenous glutathione.

Our previous work indicated that the vigor of Streptomyces decreased at the later stage of ε-poly-L-lysine (ε-PL) fermentation. In this study, we observed that the level of reactive oxygen species (ROS) in vivo increased sharply after 24 h, and the addition of an antioxidant glutathione (GSH) before this increase in ROS stimulated ε-PL synthesis in shake-flask fermentation. The enhancement of ε-PL production by GSH was further verified in batch and fed-batch fermentations. On a 5-l fermenter scale, the highest increasement was 68.8% in batch fermentation and the highest ε-PL level was 46.5 g l- 1 in fed-batch fermentation. The RT-qPCR analysis showed that the transcriptional level of the catalase gene was down-regulated, and the decrease in cell activity was alleviated by the addition of GSH. The results revealed that exogenous antioxidant might maintain the cell vigor by reducing the excess ROS which provided a novel approach to regulate ε-PL synthesis.

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.

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.

Production of ε-poly-lysine by Streptomyces albulus PD-1 via solid-state fermentation.

The aim of this study was to produce ε-poly-lysine (ε-PL) by Streptomyces albulus PD-1 through solid-state fermentation (SSF) using agro-industrial residues. Maximum ε-PL production (86.62mg/g substrate) was obtained a mixed substrate of rapeseed cake and wheat bran (2:1, w/w) supplemented with glucose (4%, w/w), (NH4)2SO4 (3%, w/w), with an initial moisture content of 65%, initial pH of 7.0 and inoculum size of 13% v/w, incubated at 30°C for 8days. The results of scanning electron microscopy indicated that the filamentous thallus could penetrate the substrate surface. Moreover, repeated-batch SSF was successfully conducted 8 times using 10% substrate as seeds for the next fermentation cycle, and the results suggest that repeated-batch SSF is more efficient because of the shortened lag phase. To the best of our knowledge, this is the first report on ε-PL production using the SSF process.

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.

Recent advances in the biotechnological production of microbial poly(ɛ-L-lysine) and understanding of its biosynthetic mechanism.

Poly(ɛ-L-lysine) (ɛ-PL) is an unusual biopolymer composed of L-lysine connected between α-carboxyl and ɛ-amino groups. It has been used as a preservative in food and cosmetics industries, drug carrier in medicines, and gene carrier in gene therapy. Modern biotechnology has significantly improved the synthetic efficiency of this novel homopoly(amino acid) on an industrial scale and has expanded its industrial applications. In the latest years, studies have focused on the biotechnological production and understanding the biosynthetic mechanism of microbial ɛ-PL. Herein, this review focuses on the current trends and future perspectives of microbial ɛ-PL. Information on the screening of ɛ-PL-producing strains, fermentative production of ɛ-PL, breeding of high-ɛ-PL-producing strains, genomic data of ɛ-PL-producing strains, biosynthetic mechanism of microbial ɛ-PL, and the control of molecular weight of microbial ɛ-PL is included. This review will contribute to the development of this novel homopoly(amino acid) and serve as a basis of studies on other biopolymers.

Effects of Chromosomal Integration of the Vitreoscilla Hemoglobin Gene (vgb) and S-Adenosylmethionine Synthetase Gene (metK) on ε-Poly-L-Lysine Synthesis in Streptomyces albulus NK660.

ε-Poly-L-lysine (ε-PL) is a widely used natural food preservative. To test the effects of the Vitreoscilla hemoglobin (VHb) and S-adenosylmethionine (SAM) on ε-PL synthesis in Streptomyces albulus NK660, the heterologous VHb gene (vgb) and SAM synthetase gene (metK) were inserted into the S. albulus NK660 chromosome under the control of the constitutive ermE* promoter. CO-difference spectrum analysis showed S. albulus NK660-VHb strain could express functional VHb. S. albulus NK660-VHb produced 26.67 % higher ε-PL and 14.57 % higher biomass than the wild-type control, respectively. Reversed-phase high-pressure liquid chromatography (RP-HPLC) results showed the overexpression of the metK gene resulted in increased intracellular SAM synthesis in S. albulus NK660-SAM, which caused increases of biomass as well as the transcription level of ε-PL synthetase gene (pls). Results indicated that the expression of vgb and metK gene improved on ε-PL synthesis and biomass for S. albulus NK660, respectively.