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Pollution caused by persistent petro-plastics is the most pressing problem currently, with 8 million tons of plastic waste dumped annually in the oceans. Plastic waste management is not systematized in many countries, because it is laborious and expensive with secondary pollution hazards. Bioplastics, synthesized by microorganisms, are viable alternatives to petrochemical-based thermoplastics due to their biodegradable nature. Polyhydroxyalkanoates (PHAs) are a structurally and functionally diverse group of storage polymers synthesized by many microorganisms, including bacteria and Archaea. Some of the most important PHA accumulating bacteria include Cupriavidus necator, Burkholderia sacchari, Pseudomonas sp., Bacillus sp., recombinant Escherichia coli, and certain halophilic extremophiles. PHAs are synthesized by specialized PHA polymerases with assorted monomers derived from the cellular metabolite pool. In the natural cycle of cellular growth, PHAs are depolymerized by the native host for carbon and energy. The presence of these microbial PHA depolymerases in natural niches is responsible for the degradation of bioplastics. Polyhydroxybutyrate (PHB) is the most common PHA with desirable thermoplastic-like properties. PHAs have widespread applications in various industries including biomedicine, fine chemicals production, drug delivery, packaging, and agriculture. This review provides the updated knowledge on the metabolic pathways for PHAs synthesis in bacteria, and the major microbial hosts for PHAs production. Yeasts are presented as a potential candidate for industrial PHAs production, with their high amenability to genetic engineering and the availability of industrial-scale technology. The major bottlenecks in the commercialization of PHAs as an alternative for plastics and future perspectives are also critically discussed.

Glycosylation can affect various protein properties such as stability, biological activity, and immunogenicity. To produce human therapeutic proteins, a host that can produce glycoproteins with correct glycan structures is required. Microbial expression systems offer economical, rapid and serum-free production and are more amenable to genetic manipulation. In this study, we developed a protocol for CRISPR/Cas9 multiple gene knockouts and knockins in Kluyveromyces marxianus, a probiotic yeast with a rapid growth rate. As hyper-mannosylation is a common problem in yeast, we first knocked out the α-1,3-mannosyltransferase (ALG3) and α-1,6-mannosyltransferase (OCH1) genes to reduce mannosylation. We also knocked out the subunit of the telomeric Ku domain (KU70) to increase the homologous recombination efficiency of K. marxianus. In addition, we knocked in the MdsI (α-1,2-mannosidase) gene to reduce mannosylation and the GnTI (β-1,2-N-acetylglucosaminyltransferase I) and GnTII genes to produce human N-glycan structures. We finally obtained two strains that can produce low amounts of the core N-glycan Man3GlcNAc2 and the human complex N-glycan Man3GlcNAc4, where Man is mannose and GlcNAc is N-acetylglucosamine. This study lays a cornerstone of glycosylation engineering in K. marxianus toward producing human glycoproteins.

Cellulosomes, which are multienzyme complexes from anaerobic bacteria, are considered nature's finest cellulolytic machinery. Thus, constructing a cellulosome in an industrial yeast has long been a goal pursued by scientists. However, it remains highly challenging due to the size and complexity of cellulosomal genes. Here, we overcame the difficulties by synthesizing the Clostridium thermocellum scaffoldin gene (CipA) and the anchoring protein gene (OlpB) using advanced synthetic biology techniques. The engineered Kluyveromyces marxianus, a probiotic yeast, secreted a mixture of dockerin-fused fungal cellulases, including an endoglucanase (TrEgIII), exoglucanase (CBHII), β-glucosidase (NpaBGS), and cellulase boosters (TaLPMO and MtCDH). The confocal microscopy results confirmed the cell-surface display of OlpB-ScGPI and fluorescence-activated cell sorting analysis results revealed that almost 81% of yeast cells displayed OlpB-ScGPI. We have also demonstrated the cellulosome complex formation using purified and crude cellulosomal proteins. Native polyacrylamide gel electrophoresis and mass spectrometric analysis further confirmed the cellulosome complex formation. Our engineered cellulosome can accommodate up to 63 enzymes, whereas the largest engineered cellulosome reported thus far could accommodate only 12 enzymes and was expressed by a plasmid instead of chromosomal integration. Interestingly, CipA 2B9C (with two cellulose binding modules, CBM) released significantly higher quantities of reducing sugars compared with other CipA variants, thus confirming the importance of cohesin numbers and CBM domain on cellulosome complex. The engineered yeast host efficiently degraded cellulosic substrates and released 3.09 g/L and 8.61 g/L of ethanol from avicel and phosphoric acid-swollen cellulose, respectively, which is higher than any previously constructed yeast cellulosome.

5–Aminolevulinic acid (5-ALA) is recently being used as a critical prodrug used for the photodynamic therapy of cancers. In this study, we first demonstrated via codon shuffling that codon adaptation index (CAI) and tRNA adaptation index (tAI) influenced the translational level and folding of the recombinant ALA synthase from Rhodobacter capsulatus (Rc*) for 5-ALA production in E. coli. A genome-based chassis Rc*GI was achieved by genomic integration and co-expression of chaperone (GroELS) with Rc*, which increased the cell growth of E. coli and 5-ALA production (up to 5.9 g/L) in antibiotic-free culture. However, the homocysteine methyltransferase (metE) and ferrienterobactin receptor (fepA), both ferric dependent and heme related proteins were stimulated after continuous culture for 7 batches, thus the addition of ferric ion into the culture boosted the cell growth and 5-ALA production. Finally, the glucose-glycerol mixed carbon source was employed to extend cell growth and the highest 5-ALA titer of 15.6 g/L was achieved via fed-batch fermentation. We provide new insights into the economical and efficient bioprocessing of 5-ALA production with a robust chassis.

Rhodotorula glutinis, an oleaginous red yeast, intrinsically produces several bio-products (i.e., lipids, carotenoids and enzymes) and is regarded as a potential host for biorefinery. In view of the limited available genetic engineering tools for this yeast, we have developed a useful genetic transformation method and transformed the β-carotene biosynthesis genes (crtI, crtE, crtYB and tHMG1) and cellulase genes (CBHI, CBHII, EgI, EgIII, EglA and BGS) into R. glutinis genome. The transformant P4-10-9-63Y-14B produced significantly higher β-carotene (27.13 ± 0.66 mg/g) than the wild type and also exhibited cellulase activity. Furthermore, the lipid production and salt tolerance ability of the transformants were unaffected. This is the first study to engineer the R. glutinis for simultaneous β-carotene and cellulase production. As R. glutinis can grow in sea water and can be engineered to utilize the cheaper substrates (i.e. biomass) for the production of biofuels or valuable compounds, it is a promising host for biorefinery.

In this study, an astaxanthin-biosynthesis Kluyveromyces marxianus strain Sm23 was first constructed, which could produce 31µg/g DCW astaxanthin. Then, repeated genome integration of the key astaxanthin biosynthesis genes Hpchyb and bkt was done to increase gene copy number and astaxanthin yield. Four improved strains were obtained and the yield of astaxanthin and the total yield of carotenoids in a strain increased with the copy numbers of Hpchyb and bkt. To improve the yield further, the gene Hpchyb from Haematococcus pluvialis was modified by site-directed mutagenesis to increase the enzyme efficiency or/and to prevent the heterologous protein degradation by ubiquitination. Using repeated-integration approach of bkt and the mutated Hpchyb into Sm23, the S3-2 strain was obtained and shown to produce the 3S, 3'S-astaxanthin at 9972µg/g DCW in a 5L fermentor.

Background A microorganism engineered for non-native tasks may suffer stresses it never met before. Therefore, we examined whether a Kluyveromyces marxianus strain engineered with a carotenoid biosynthesis pathway can serve as an anti-stress chassis for building cell factories. Results Carotenoids, a family of antioxidants, are valuable natural products with high commercial potential. We showed that the free radical removal ability of carotenoids can confer the engineered host with a higher tolerance to ethanol, so that it can produce more bio-ethanol than the wild type. Moreover, we found that this engineered strain has improved tolerance to other toxic effects including furfurals, heavy metals such as arsenate (biomass contaminant) and isobutanol (end product). Furthermore, the enhanced ethanol tolerance of the host can be applied to bioconversion of a natural medicine that needs to use ethanol as the delivery solvent of hydrophobic precursors. The result suggested that the engineered yeast showed enhanced tolerance to ethanol-dissolved hydrophobic 10-deacetylbaccatin III, which is considered a sustainable precursor for paclitaxel (taxol) bioconversion. Conclusions The stress tolerances of the engineered yeast strain showed tolerance to several toxins, so it may serve as a chassis for cell factories to produce target products, and the co-production of carotenoids may make the biorefinary more cost-effective.

Background Enzymatic conversion of lignocellulosic biomass into soluble sugars is a major bottleneck in the plant biomass utilization. Several anaerobic organisms cope these issues via multiple-enzyme complex system so called ‘cellulosome’. Hence, we proposed a “biomimic operon” concept for making an artificial cellulosome which can be used as a promising tool for the expression of cellulosomal enzymes in Bacillus subtilis. Results According to the proteomic analysis of Clostridium thermocellum ATCC27405 induced by Avicel or cellobiose, we selected eight highly expressed cellulosomal genes including a scaffoldin protein gene (cipA), a cell-surface anchor gene (sdbA), two exoglucanase genes (celK and celS), two endoglucanase genes (celA and celR), and two xylanase genes (xynC and xynZ). Arranging these eight genes in two different orders, we constructed two different polycistronic operons using the ordered gene assembly in Bacillus method. This is the first study to express the whole CipA along with cellulolytic enzymes in B. subtilis. Each operon was successfully expressed in B. subtilis RM125, and the protein complex assembly, cellulose-binding ability, thermostability, and cellulolytic activity were demonstrated. The operon with a higher xylanase activity showed greater saccharification on complex cellulosic substrates such as Napier grass than the other operon. Conclusions In this study, a strategy for constructing an efficient cellulosome system was developed and two different artificial cellulosomal operons were constructed. Both operons could efficiently express the cellulosomal enzymes and exhibited cellulose saccharification. This strategy can be applied to different industries with cellulose-containing materials, such as papermaking, biofuel, agricultural compost, mushroom cultivation, and waste processing industries.

Cellulose is a renewable feedstock for green industry. It is therefore important to develop a technique to construct a host with a high cellulolytic efficiency to digest cellulose. In this study, we developed a convenient host-engineering technique to adjust the expression levels of heterologous genes in the host by promoter rearrangement and gene copy number adjustment. Using genes from different glycoside hydrolase (GH) families including GH2, GH3, GH5, GH6, GH7, and GH12 from Aspergillus niger, Trichoderma reesei, and Neocallimastix patriciarum, we constructed a cellulolytic Kluyveromyces marxianus with eight cellulase gene-cassettes that produced a cellulase cocktail with a high cellulolytic efficiency, leading to a significant reduction in enzyme cost in a rice straw saccharification process. Our technique can be used to design a host that can efficiently convert biomass feedstock to biofuel.

A novel dual-microbe Bacillus/yeast co-culture system is developed for cellulosic bioethanol production. A recombinant cellulosomal Bacillus subtilis that carries eight cellulosomal genes of Clostridium thermocellum, including one scaffolding protein gene (cipA), one cell-surface anchor gene (sdbA), two exo-glucosidase genes (celK and celS), two endoglucanase genes (celA and celR), and two xylanase genes (xynC and xynZ) was constructed. The partner microbes for the dual-microbe combination are the wild type kefir yeast Kluyveromyces marxianus KY3, K. marxianus KY3-NpaBGS, which carries a β-glucosidase (NpaBGS) gene from rumen fungus, and the K. marxianus KR5 strain, that harbors endoglucanase (egIII), exo-glucanase (cbhI) and NpaBGS genes. All three Bacillus/yeast co-culture systems could achieve the cellulose saccharification and ethanol conversion simultaneously better than KR5 alone. The combination of Bacillus/KY3-NpaBGS outperformed that of Bacillus/KY3, as they could produce β-glucosidase enzyme for the system. Although, KR5 produces two more kinds of cellulases than KY3-NpaBGS. Bacillus/KR5 could not perform better than Bacillus/KY3-NpaBGS. Our results suggest that the dual-microbe Bacillus/yeast co-culturing system could leverage the advantages from both microbes and have a great potential for integrating into consolidated bioprocessing system.

In order to enhance anaerobic hydrogen production from yeast waste, a series of 120-mL batch co-cultures of Clostridium beijerinckii L9, Clostridium butyricum M1, and Bacillus thermoamylovorans B5 under mesophilic conditions were established according to full factorial design (FFD) and mixture design (MD). The experimental results were subjected to multivariate and response surface analyses to determine the relationships between bacteria converting yeast waste into hydrogen. The results indicated clearly that C. beijerinckii L9 and C. butyricum M1 had significant potential to convert yeast waste into hydrogen. There was no significant hydrogen generation when B. thermoamylovorancs B5 alone was cultured with yeast waste. However, B. thermoamylovorancs B5 could significantly shorten the co-culture’s hydrogen-producing lag phase. Response surface analyses demonstrate that B. thermoamylovorancs B5 can stimulate the specific hydrogen production rate of C. beijerinckii L9 and C. butyricum M1, greater in the case of the former than of the latter. An ultimate hydrogen yield of 46 mL H 2 /g COD added yeast waste was obtained with an optimal volumetric ratio C. beijerinckii L9: C. butyricum M1: B. thermoamylovoranc B5 of 8.9:4.8:10.3. Highly reproducible co-culture results confirm that FFD and MD, via response surface analysis, are applicable to assess the roles of the individual microorganisms in the defined co-culture.

For simultaneous saccharification and fermentation of cellulosic bioresources to biofuels, a stable rumen-mimicking bacterial consortium was established and maintained by sub-culturing and is referred to here as the “Functional Rumen Bacterial Consortium” (FRBC). The microbial community was analyzed by denaturing gradient gel electrophoresis, which contained dominant species such as Clostridium xylanolyticum, Clostridium papyrosolvens, Clostridium beijerinckii, Clostridium puniceum, Clostridium putrifaciens, and Ruminococcus sp. These predominant Clostridial strains showed potential in converting lignocellulosic material into bio-hydrogen and bio-fuels. The Clostridial strains with 16S rRNA sequences identical to the DGGE profile were isolated from FRBC. The isolate C. puniceum strain Ru6 exhibited xylanase and pectinase activity and higher hydrogen productivity and was thus chosen as the partner strain for ethanol production with C. xylanolyticum Ru15, which showed additional endoglucanase activity. A co-culture approach was used to reconstruct the FRBC with Ru6 and Ru15, and the productivities of hydrogen and ethanol were comparable to that of the FRBC. These results demonstrate the potential of a biologically inspired rumen-mimic microbial community to produce cellulosic bio-fuels.

Using anaerobic micro-organisms to convert organic waste to produce hydrogen gas gives the benefits of energy recovery and environmental protection. The objective of this study was to develop a biohydrogen production technology from food wastewater focusing on hydrogen production efficiency and micro-flora community at different hydraulic retention times. Soluble condensed molasses fermentation (CMS) was used as the substrate because it is sacchariferous and ideal for hydrogen production. CMS contains nutrient components that are necessary for bacterial growth: microbial protein, amino acids, organic acids, vitamins and coenzymes. The seed sludge was obtained from the waste activated sludge from a municipal sewage treatment plant in Central Taiwan. This seed sludge was rich in Clostridium sp. A CSTR (continuously stirred tank reactor) lab-scale hydrogen fermentor (working volume, 4.0 L) was operated at a hydraulic retention time (HRT) of 3–24 h with an influent CMS concentration of 40 g COD/L. The results showed that the peak hydrogen production rate of 390 mmol H 2 /L-d occurred at an organic loading rate (OLR) of 320 g COD/L-d at a HRT of 3 h. The peak hydrogen yield was obtained at an OLR of 80 g COD/L-d at a HRT of 12 h. At HRT 8 h, all hydrogenase mRNA detected were from Clostridium acetobutylicum-like and Clostridium pasteurianum -like hydrogen-producing bacteria by RT-PCR analysis. RNA based hydrogenase gene and 16S rRNA gene analysis suggests that Clostridium exists in the fermentative hydrogen-producing system and might be the dominant hydrogen-producing bacteria at tested HRTs (except 3 h). The hydrogen production feedstock from CMS is lower than that of sucrose and starch because CMS is a waste and has zero cost, requiring no added nutrients. Therefore, producing hydrogen from food wastewater is a more commercially feasible bioprocess.

The study aimed to establish stable rumen-mimic bacterial consortia as a functional union for simultaneous saccharification and fermentation from cellulosic bioresource. The consortia was constructed by repeated-batch culture with ruminal microflora and napiergrass at 38 °C. The major bacterial composition of batch culture was monitored by 16S rRNA gene-targeted denaturing gradient gel electrophoresis (DGGE). The result showed that a stable consortia constituted by ruminal microflora was formed, and the consortia includes bacterial strains such as Clostridium xylanolyticum, Clostridium papyrosolvens, Clostridium beijerinckii, Ruminococcus sp., Ethanoligenens harbinense, and Desulfovibrio desulfuricans. The Clostridium genus was showed as the dominant population in the system and contributed to the biohydrogen production. During each eight days incubation period, the functional consortia could degrade an average of 27% hemicellulose and 2% cellulose from napiergrass biomass. While the increasing of the reducing sugars and their converting to biohydrogen gas productivity were also observed. The time course profile for cellulytic enzymes showed that the hydrolysis of complex lignocellulosic material may occur through the ordered actions of xylenase and cellulase activities.

An anaerobic continuous-flow hydrogen fermentor was operated at a hydraulic retention time of 8 h using condensed molasses fermentation solubles (CMS) substrate of 40 g-COD/L. Serum bottles were used for seed micro-flora cultivation and batch hydrogen fermentation tests (CMS substrate concentrations of 10–160 g-COD/L). Three hydrogen-producing bacterial strains Clostridium sporosphaeroides F52, Clostridium tyrobutyricum F4 and Clostridium pasteurianum F40 were isolated from the seed fermentor and used as the seeding microbes in single and mixed-culture cultivations for determining their hydrogen productivity. These strains possessed specific hydrogenase genes that could be detected from CMS-fed hydrogen fermentors and were major hydrogen producers. C. pasteurianum F40 was the dominant strain with a high hydrogen production rate while C. sporosphaeroides F52 may play a main role in degrading carbohydrate and glutamate. These strains could be co-cultivated as a symbiotic mixed-culture process to enhance hydrogen productivity. C. pasteurianum F40 or C. tyrobutyricum F4 co-culture with the glutamate-utilizing strain C. sporosphaeroides F52 efficiently enhanced hydrogen production by 12–220% depending on the substrate CMS concentrations.

By using brewery yeast waste and microflora from rice straw compost, an anaerobic semi-solid bio-hydrogen-producing system has been established. For the purpose of industrialization, the major players of both aerobic and anaerobic bacterial strains in the system were isolated and their combination for an effective production of bio-hydrogen and other bio-fuels was examined in this study. The phylogenetic analysis found that four anaerobic isolates (Clostridium beijerinckii L9, Clostridium diolis Z2, Clostridium roseum Z5-1, and C. roseum W8) were highly related with each other and belongs to the cluster I clostridia family, the family that many of solvent-producing strains included. On the other hand, one of the aerobic isolates, the Bacillus thermoamylovorans strain I, shown multiple extracellular enzyme activities including lipase, protease, α-amylase, pectinase and cellulase, was suggested as a good partner for creating an anaerobic environment and pre-saccharification of substrate for those co-cultured solventogenic clostridial strain. Among these clostridial strains, though C. beijerinckii L9 do not show as many extracellular enzyme activities as Bacillus, but it performs the highest hydrogen-producing ability. The original microflora can be updated to a syntrophic bacterial co-culture system contended only with B. thermoamylovorans I and C. beijerinckii L9. The combination of aerobic Bacillus and anaerobic Clostridium may play the key role for developing the industrialized bio-fuels and bio-hydrogen-producing system from biomass.

An anaerobic continuous-flow fermentation system with condensed molasses fermentation soluble (CMS) from monosodium glutamate production as substrate was developed for the biohydrogen production. The production rate of the system was increased, while hydraulic retention time (HRT) was varied from 12 to 3 h and the optimal hydrogen production rate was obtained at HRT 3 h. The major bacterial composition of each HRT was analyzed by 16S rRNA gene-targeted denaturing gradient gel electrophoresis (DGGE). Acidaminococcus sp. that could evolve hydrogen from glutamate was the only hydrogen producer and could be also detected from substrate. Hydrogenase mRNA-targeted reverse transcriptase polymerase chain reaction (RT-PCR) analysis showed that except HRT 3 h, two clusters of clostridial hydrogenase gene sequences were obtained. RT-PCR-DGGE analysis was employed to illustrate the active clostridial strains confirming that clostridial strains were not present at HRT 3 h. Acidaminococcus sp. existing in substrate might be the major strain that contributes to hydrogen production at HRT 3 h.

The algal β-carotene hydroxylase gene Crchyb from Chlamydomonas reinhardtii, Czchyb from Chlorella zofingiensis, or Hpchyb from Haematococcus pluvialis and six other carotenoid-synthesis pathway genes were co-integrated into the genome of a yeast host. Each of these three algal genes showed a higher efficiency to convert β-carotene to downstream carotenoids than the fungal genes from Phaffia rhodozyma. Furthermore, the strain with Hpchyb displayed a higher carotenoid productivity than the strains integrated with Crchyb or Czchyb, indicating that Hpchyb is more efficient than Crchyb and Czchyb. These results suggest that β-carotene hydroxylase plays a crucial role in the biosynthesis of carotenoids.

Background Though n-butanol has been proposed as a potential transportation biofuel, its toxicity often causes oxidative stress in the host microorganism and is considered one of the bottlenecks preventing its efficient mass production. Results To relieve the oxidative stress in the host cell, metallothioneins (MTs), which are known as scavengers for reactive oxygen species (ROS), were engineered in E. coli hosts for both cytosolic and outer-membrane-targeted (osmoregulatory membrane protein OmpC fused) expression. Metallothioneins from human (HMT), mouse (MMT), and tilapia fish (TMT) were tested. The host strain expressing membrane-targeted TMT showed the greatest ability to reduce oxidative stresses induced by n-butanol, ethanol, furfural, hydroxymethylfurfural, and nickel. The same strain also allowed for an increased growth rate of recombinant E. coli under n-butanol stress. Further experiments indicated that the TMT-fused OmpC protein could not only function in ROS scavenging but also regulate either glycine betaine (GB) or glucose uptake via osmosis, and the dual functional fusion protein could contribute in an enhancement of the host microorganism’s growth rate. Conclusions The abilities of scavenging intracellular or extracellular ROS by these engineering E. coli were examined, and TMT show the best ability among three MTs. Additionally, the membrane-targeted fusion protein, OmpC-TMT, improved host tolerance up to 1.5% n-butanol above that of TMT which is only 1%. These results presented indicate potential novel approaches for engineering stress tolerant microorganism strains.

Background Cellulose, which is the most abundant renewable biomass on earth, is a potential bio-resource of alternative energy. The hydrolysis of plant polysaccharides is catalyzed by microbial cellulases, including endo-β-1,4-glucanases, cellobiohydrolases, cellodextrinases, and β-glucosidases. Converting cellobiose by β-glucosidases is the key factor for reducing cellobiose inhibition and enhancing the efficiency of cellulolytic enzymes for cellulosic ethanol production. Results In this study, a cDNA encoding β-glucosidase was isolated from the buffalo rumen fungus Neocallimastix patriciarum W5 and is named NpaBGS. It has a length of 2,331 bp with an open reading frame coding for a protein of 776 amino acid residues, corresponding to a theoretical molecular mass of 85.1 kDa and isoelectric point of 4.4. Two GH3 catalytic domains were found at the N and C terminals of NpaBGS by sequence analysis. The cDNA was expressed in Pichia pastoris and after protein purification, the enzyme displayed a specific activity of 34.5 U/mg against cellobiose as the substrate. Enzymatic assays showed that NpaBGS was active on short cello-oligosaccharides from various substrates. A weak activity in carboxymethyl cellulose (CMC) digestion indicated that the enzyme might also have the function of an endoglucanase. The optimal activity was detected at 40°C and pH 5 ~ 6, showing that the enzyme prefers a weak acid condition. Moreover, its activity could be enhanced at 50°C by adding Mg2+ or Mn2+ ions. Interestingly, in simultaneous saccharification and fermentation (SSF) experiments using Saccharomyces cerevisiae BY4741 or Kluyveromyces marxianus KY3 as the fermentation yeast, NpaBGS showed advantages in cell growth, glucose production, and ethanol production over the commercial enzyme Novo 188. Moreover, we showed that the KY3 strain engineered with the NpaNGS gene can utilize 2 % dry napiergrass as the sole carbon source to produce 3.32 mg/ml ethanol when Celluclast 1.5 L was added to the SSF system. Conclusion Our characterizations of the novel β-glucosidase NpaBGS revealed that it has a preference of weak acidity for optimal yeast fermentation and an optimal temperature of ~40°C. Since NpaBGS performs better than Novo 188 under the living conditions of fermentation yeasts, it has the potential to be a suitable enzyme for SSF.