Efficient poly(ß-L-malic acid) production from cassava hydrolysate by cell recycle of Aureobasidium pullulans.

Poly(ß-L-malic acid) (PMLA) is a water-soluble, biodegradable, and biocompatible polymer with broad prospective applications and can be hydrolyzed to produce widely used acidulant L-malic acid. In order to meet an increasing demand of PMLA, we employed two effective cell-recycling strategies to produce PMLA from raw cassava hydrolysate by Aureobasidium pullulans ZD-3d. In fed-batch fermentation with raw cassava hydrolysate, 101.9 g/L PMLA was obtained with the productivity and yield of 0.77 g/L/h and 0.40 g/g, respectively. Further, three times of membrane filtration-based cell recycling fermentation was carried out, with a high productivity and yield of 1.04-1.64 g/L/h and 0.5-0.84 g/g achieved, respectively. While harnessing centrifugation-based cell recycling fermentation for five times, the productivity and yield approached 0.98-1.76 g/L/h and 0.78-0.86 g/g, respectively. To our knowledge, the processes showed the highest average PMLA productivity compared with others using low-cost biomass, which offered efficient and economical alternatives for PMLA production. KEY POINTS: • PMLA production from raw cassava hydrolysate by Aureobasidium pullulans was studied • High PMLA productivity and yield were obtained via two cell recycling strategies • The highest average PMLA productivity from low-cost biomass to date was achieved.

Poly(ß-L-malic acid) (PMLA) from Aureobasidium spp. and its current proceedings.

Poly(ß-L-malic acid) is one natural biopolymer that has the outstanding features of biocompatibility, biodegradability, water solubility, and non-immunogenicity, and it is easily chemically modified. So poly(ß-L-malic acid) (PMLA) and its derivatives may have a great potential application as a novel drug delivery system and in the production of advanced biomaterials which have attracted so much research attention. The fungi of Aureobasidium spp. have been discovered to be the most suitable candidates for PMLA production in large quantities which satisfy the demand of either research or industry. In this review, we will give an overall summary about the PMLA produced by Aureobasidium spp. based on related research in the last decades and the elaboration of this PMLA producer will also be accomplished. More importantly, the latest proceedings will be specified and some suggestions to the elucidation of a PMLA biosynthesis pathway which remains undefined up to date will be proposed. Finally, through this review, the further exploitation for the application of PMLA from Aureobasidium spp. can be emphasized and promoted.

Taxonomy of Aureobasidium spp. and biosynthesis and regulation of their extracellular polymers.

The genus Aureobasidium spp. have been divided into three species, A. pullulans. A. leucospermi and A. proteae, and A. pullulans has been known to have five varieties. However, after analysis of many strains of this yeast isolated from different environments, they do not belong to any of the three species or the five varieties. Although pullulan produced by A. pullulans has been widely used in different fields in industry and different strains of this yeast has been known to produce poly(ß-L-malic acid) (PMA), heavy oils and ß-1,3-glucan, it is still unknown how the black yeast synthesizes and secretes the extracellular polymers at molecular level. In this review article, new biosynthetic pathways of pullulan, PMA and heavy oils, the enzymes and their genes related to their biosynthesis and regulation are proposed. Furthermore, some enzymes and their genes related to pullulan biosynthesis in A. pullulans have been characterized. But it is completely unknown how pullulan is secreted and how PMA, heavy oils and ß-1,3-glucan are synthesized and secreted. Therefore, there is much work to be done about taxonomy and biosynthesis, secretion and regulation of pullulan, PMA, heavy oils and ß-1,3-glucan at molecular levels in Aureobasidium spp.

Biosynthesis of poly(ß-L-malic acid) from rubberwood enzymatic hydrolysates in co-fermentation by Aureobasidium pullulans.

Co-fermentation of multiple substrates has emerged as the most effective method to improve the yield of bioproducts. Herein, sustainable rubberwood enzymatic hydrolysates (RWH) were co-fermented by Aureobasidium pullulans to produce poly(ß-L-malic acid) (PMA), and RWH + glucose/xylose was also investigated as co-substrates. Owing to low inhibitor concentration and abundant natural nitrogen source content of RWH, a high PMA yield of 0.45 g/g and a productivity of 0.32 g/L/h were obtained by RWH substrate fermentation. After optimization, PMA yields following the fermentation of RWH + glucose and RWH + xylose reached 59.92 g/L and 53.71 g/L, respectively, which were 52 % and 36 % higher than that after the fermentation of RWH. RWH + glucose more significantly affected the correlation between PMA yield and substrate concentration than RWH + xylose. The results demonstrated that the co-fermentation of RWH co-substrate is a promising method for the synthesis of bioproducts.

Fractionation and characterization of poly(ß-L-malic acid) produced by Aureobasidium melanogenum ipe-1.

Poly (ß-L-malic acid) (PMLA) is attracting industrial interest for its potential application in medicine and other industries, whose functions primarily depend upon its molecular size and chemical structure. Up to now, the fractionation and characterization of PMLA produced by Aureobasidium spp. were still unclear. In this study, the product from A. melanogenum ipe-1 was effectively fractionated using 300 and 50 kDa membranes. During the filtration, the mechanisms of membrane fouling were illegible since the PMLA can both reject and permeate the membrane, while the main fouling mechanism varied between standard blocking and complete blocking during the diafiltration. After fractionation, 14.0, 8.4 and 77.6 % of the PMLAs with Mws of 75,134, 21,344 and 10,056 Da were distributed in the 300 kDa retentate after diafiltrating, 50 kDa retentate after diafiltrating, and the 50 kDa permeate, respectively. The Mw/Mns of the PMLAs were 4.12, 1.92, and 1.12 in the three fractions. Based on characteristic spectra of NMR, HPLC and FTIR, the product was not usual L-malic acid monomers, but glucose-terminated PMLA. The glucose was located at the terminal hydroxyl of PMLA. These results would serve as a valuable guide for process design and practical operation in subsequent industrial application.

Green process for isolation and purification of poly(ß-L-malic acid) from Aureobasidium spp. by an integrated ion exchange and membrane separation.

Poly (ß-L-malic acid) (PMLA) is a biopolymer used in food and medical fields. However, the industrial processes are susceptible to the pollution of CaSO4 waste and organic solvent owing to the heavy use of CaCO3 in fermentation process and organic solvents in isolation process. This study developed an organic solvent and CaSO4 -free process for the industrial-scale production of PMLA. Firstly, calcium ion was removed at pH 9.2 by pH adjustment with Na2CO3, and the generated CaCO3 was reused in the fermentation process. Then, the D296 resin was selected to isolate the PMLA from the Ca2+-free broth, where the adsorption data were both primely described by the Freundlich and Langmuir equation, while Freundlich model better fit the process than Langmuir equation, indicating that it was non-monolayer adsorption of PMLA on the resin. Meanwhile, a three-step gradient elution with phosphate buffer (i.e., 0.2 mol/L, pH 7.0) containing 0.1, 0.2 and 1 mol/L NaCl was developed to recover PMLA. Finally, a PES15 membrane was selected to recover the PMLA from the elution solution, which could be reused in the next cycle. As a result, the PMLA with a purity of 98.89 % was obtained with the developed green process. In the developed process, it removed the pollution of organic solvent and calcium waste for the biosynthesis of PMLA on an industrial scale, which also offers a sustainable and green route for the biosynthesis of other carboxylic acids.

Electrolytic stimulation in aid of poly(ß-L-malic acid) production by Aureobasidium melanogenum ipe-1.

Poly (ß-L-malic acid) (PMLA) is attracting industrial interest for its potential application in medicine and other industries. In this study, electrolytic stimulation assisted PMLA production was developed. Firstly, it was found that the pentavalent nitrogen source (i.e., NO3-) was more suitable for PMLA production. Secondly, a usual single-chamber bioelectric-fermentation system (BES) cannot improve PMLA production, which can only promote cell growth. Then, a new single-chamber BES with an external circulation was developed, where the PMLA metabolism was further intensified. Finally, the integration of NO3- addition and electrolytic stimulation mode (c) showed a positive synergy on the PMLA production. Compared to the case without NO3- addition and electrolytic stimulation, the PMLA production was increased by 22.9 % using the integrated process. Moreover, compared to the case without the electrolytic stimulation mode (c), it was revealed that the different genes involved in 12 metabolic subsystems using the integrated process, where 31 and 177 genes were up-regulated and down-regulated, respectively. The up-regulated genes were mainly participated in melanin metabolic process, catalase activity, and oxidoreductase activity. Hence, the integration of electrolytic stimulation represents a novel approach to improve PMLA production.

Small-Sized Co-Polymers for Targeted Delivery of Multiple Imaging and Therapeutic Agents.

Research has increasingly focused on the delivery of high, often excessive amounts of drugs, neglecting negative aspects of the carrier's physical preconditions and biocompatibility. Among them, little attention has been paid to "small but beautiful" design of vehicle and multiple cargo to achieve effortless targeted delivery into deep tissue. The design of small biopolymers for deep tissue targeted delivery of multiple imaging agents and therapeutics (mini-nano carriers) emphasizes linear flexible polymer platforms with a hydrodynamic diameter of 4 nm to 10 nm, geometrically favoring dynamic juxtaposition of ligands to host receptors, and economic drug content. Platforms of biodegradable, non-toxic poly(ß-l-malic acid) of this size carrying multiple chemically bound, optionally nature-derived or synthetic affinity peptides and drugs for a variety of purposes are described in this review with specific examples. The size, shape, and multiple attachments to membrane sites accelerate vascular escape and fast blood clearance, as well as the increase in medical treatment and contrasts for tissue imaging. High affinity antibodies routinely considered for targeting, such as the brain through the blood-brain barrier (BBB), are replaced by moderate affinity binding peptides (vectors), which penetrate at high influxes not achievable by antibodies.

Poly(malic acid) production from liquefied corn starch by simultaneous saccharification and fermentation with a novel isolated Aureobasidium pullulans GXL-1 strain and its techno-economic analysis.

In this study, a novel Aureobasidium pullulans GXL-1 strain without melanin secretion was isolated for efficient polymalic acid (PMA) production. The PMA produced by GXL-1 was characterized, and its molecular mass was determined to be 1.621 kDa by gel permeation chromatography. Liquefied corn starch was shown to replace glucose for PMA production by GXL-1 through simultaneous saccharification and fermentation. The PMA titer obtained from batch fermentation was up to 49.0 ± 1.6 g/L in a 10 L fermentor, and the PMA yield and productivity obtained from repeated-batch fermentation were up to 0.50 g/g and 0.34 g/L·h, respectively. Furthermore, process design and techno-economic analysis were performed at an annual output level of 5000 metric tons by SuperPro Designer. Results showed that the production cost of $2.046/kg and payback period of 6.9 years were achieved by repeated-batch fermentation; this provides an economically feasible strategy for industrial-scale production of PMA.

A novel PMA synthetase is the key enzyme for polymalate biosynthesis and its gene is regulated by a calcium signaling pathway in Aureobasidium melanogenum ATCC62921.

It has been well known that poly(ß-l-malic acid)(PMA) has many potential applications. However, it is still completely unknown how PMA is biosynthesized in Aureobasidium spp. In this study, it was found that malic acid from TCA cycle was the main source for PMA biosynthesis. Especially, the novel PMA synthetase encoded by the PMAs gene, a non-ribosomal peptide synthetase (NRPS) containing A like domain, T domain and C like domain was the key enzyme for polymerization of malate into PMA. Therefore, abolishment of the PMAs gene encoding the novel PMA synthetase rendered the mutant ΔPMAs-3 totally to lose the ability to synthesize any PMA and complementation of the PMAs gene partially restored PMA biosynthesis, but the mutant could grow normally on the YPD plate and in the PMA medium with CaCO3. The transcriptional activator Crz1 in the Ca2+-signal pathway controlled expression of the PMAs gene and PMA biosynthesis. The complete elucidation of the PMA biosynthesis pathway and its regulation was of significant for a deeper understanding of detailed yeast-like fungal PMA synthesis, metabolic engineering and molecular editing for modifying PMA biosynthesis and its physicochemical properties.

Development and benefit evaluation of fermentation strategies for poly(malic acid) production from malt syrup by Aureobasidium melanogenum GXZ-6.

Malt syrup, as a low-cost substrate without any pretreatment, was proved to be able to replace maltose for ploymalic acid (PMA) production by Aureobasidium melanogenum GXZ-6. The PMA titer of 55.53 ±â€¯1.72 g/L was obtained by batch fermentation in a 10-L fermentor with addition of malate, citrate and sodium malonate. Then, a higher PMA titer of 124.07 ±â€¯2.28 g/L was obtained in fed-batch fermentation, which increased by 123.43% than that from batch fermentation. Moreover, repeated-batch fermentation with three batches gave a PMA titer of 64.06 g/L on average with a higher yield of 0.81 g/g and productivity of 0.56 g/L·h. Fermentation process and economics analysis were performed by SuperPro Designer for a 2000 metric tons plant. Results showed that PMA production cost was as low as $ 1.716/kg by fed-batch fermentation, which provides an economical strategy for large-scale PMA production.

Analysis of the L-malate biosynthesis pathway involved in poly(ß-L-malic acid) production in Aureobasidium melanogenum GXZ-6 by addition of metabolic intermediates and inhibitors.

Poly(ß-L-malic acid) (PMA) is a promising polyester formed from L-malate in microbial cells. Malate biosynthesis is crucial for PMA production. Previous studies have shown that the non-oxidative pathway or oxidative pathway (TCA cycle) is the main biosynthetic pathway of malate in most of PMA-producing strains, while the glyoxylate cycle is only a supplementary pathway. In this study, we investigated the effect of exogenous metabolic intermediates and inhibitors of the malate biosynthetic pathway on PMA production by Aureobasidium melanogenum GXZ-6. The results showed that PMA production was stimulated by maleic acid (a fumarase inhibitor) and sodium malonate (a succinate dehydrogenase inhibitor) but inhibited by succinic acid and fumaric acid. This indicated that the TCA cycle might not be the only biosynthetic pathway of malate. In addition, the PMA titer increased by 18.1% upon the addition of glyoxylic acid after 72 h of fermentation, but the PMA titer decreased by 7.5% when itaconic acid (an isocitrate lyase inhibitor) was added, which indicated that malate for PMA production was synthesized significantly via the glyoxylate cycle rather than the TCA cycle. Furthermore, in vitro enzyme activities of the TCA and glyoxylate cycles suggested that the glyoxylate cycle significantly contributed to the PMA production, which is contradictory to what has been reported previously in other PMA-producing A. pullulans.

Production of poly(ß-l-malic acid) by Aureobasidium pullulans HA-4D under solid-state fermentation.

Poly(ß-l-malic acid) (PMA) production by Aureobasidium pullulans HA-4D was carried out through solid-state fermentation (SSF) using agro-industrial residues. Maximum PMA production (75.4mg/g substrate) was obtained from a mixed substrate of sweet potato residue and wheat bran (1:1, w/w) supplemented with NaNO3 (0.8%, w/w) and CaCO3 (2%, w/w), with an initial moisture content of 70% and inoculum size of 13% (v/w) for 8days. Repeated-batch SSF was successfully conducted for 5 cycles with a high productivity. The scanning electron microscopy showed that the yeast-like cells of A. pullulans HA-4D could grow well on the solid substrate surface. Moreover, the cost analysis showed that the unit price of PMA in SSF was much lower than that of SmF. This is the first report on PMA production via SSF, and this study provided a new method to produce PMA from inexpensive agro-industrial residues.