Engineering an (R)-selective transaminase for asymmetric synthesis of (R)-3-aminobutanol.

(R)-selective transaminases show promise as catalysts for the asymmetric synthesis of chiral amines, which are building blocks of various small molecule drugs. However, their application is limited by poor substrate acceptance and low catalytic efficiency. Here, a potential (R)-selective transaminase from Fodinicurvata sediminis (FsTA) was identified through a substrate truncating strategy, and used as starting point for enzyme engineering toward catalysis of 4-hydroxy-2-butanone, a substrate that poses challenges in catalysis. Molecular docking and dynamics simulations revealed Y90 as the key residue responsible for poor substrate binding. Starting from the variant (Y90F, mut1) with initial activity, FsTA was systematically modified to improve substrate-binding through active site reshaping and consensus sequence strategy, yielding three variants (H30R, V152K, and Y156F) with improved activity. A quadruple mutation variant H30R/Y90F/V152K/Y156F (mut4) was also found to show a 7.95-fold greater catalytic efficiency (kcat/KM) than the initial variant mut1. Furthermore, mut4 also enhanced the thermostability of enzyme significantly, with the Tm value increasing by 10 °C. This variant also exhibited significantly improved activity toward a series of ketones that are either not accepted or poorly accepted by the wild-type. This study provides a basis for the rational design of an active to creating variants that can accommodate novel substrates.

[Development and exploration of the national first-class undergraduate course in "Enzyme Engineering"].

The biotechnology industry is a strategic emerging industry in our country, holding a crucial position in the national economy. The training of innovative high-quality professionals carries immense significance. As the cornerstone course in biotechnology, "Enzyme Engineering" directly impacts the students' caliber and industry development. This course aims to address pertinent issues present in the current curriculum delivery, such as inadequately optimized content, excessive dependency on textbooks, and reliance on monotonous teaching methods. By adjusting the course outline, updating the case material repository, and engaging students' enthusiasm, we developed a three-dimensional approach to instruct. This approach included a blend of online and offline components, interactive teaching through the flipped classroom methodology, heuristic teaching using problem-based learning (PBL) mode, and topic teaching via case studies. We also improved the assessment mechanism to stimulate students' enthusiasm for learning nurture their innovation capabilities. Our objective was to foster high-quality professionals with a robust foundation and practical expertise. Through teaching exploration and practice, we have witnessed significant improvement in both teaching efficacy and students' engineering practice and innovation abilities.

Computer-aided rational design strategy based on protein surface charge to improve the thermal stability of a novel esterase from Geobacillus jurassicus.

OBJECTIVES: Although Geobacillus are significant thermophilic bacteria source, there are no reports of thermostable esterase gene in Geobacillus jurassicus or rational design strategies to increase the thermal stability of esterases. RESULTS: Gene gju768 showed a highest similarity of 15.20% to esterases from Geobacillus sp. with detail enzymatic properties. Using a combination of Gibbs Unfolding Free Energy (∆∆G) calculator and the distance from the mutation site to the catalytic site (DsCα-Cα) to screen suitable mutation sites with elimination of negative surface charge, the mutants (D24N, E221Q, and E253Q) displayed stable mutants with higher thermal stability than the wild-type (WT). Mutant E253Q exhibited the best thermal stability, with a half-life (T1/2) at 65 °C of 32.4 min, which was 1.8-fold of the WT (17.9 min). CONCLUSION: Cloning of gene gju768 and rational design based on surface charge engineering contributed to the identification of thermostable esterase from Geobacillus sp. and the exploration of evolutionary strategies for thermal stability.

CRISPR/Cas9-based toolkit for rapid marker recycling and combinatorial libraries in Komagataella phaffii.

Komagataella phaffii, a nonconventional yeast, is increasingly attractive to researchers owing to its posttranslational modification ability, strict methanol regulatory mechanism, and lack of Crabtree effect. Although CRISPR-based gene editing systems have been established in K. phaffii, there are still some inadequacies compared to the model organism Saccharomyces cerevisiae. In this study, a redesigned gRNA plasmid carrying red and green fluorescent proteins facilitated plasmid construction and marker recycling, respectively, making marker recycling more convenient and reliable. Subsequently, based on the knockdown of Ku70 and DNA ligase IV, we experimented with integrating multiple DNA fragments at a single locus. A 26.5-kb-long DNA fragment divided into 11 expression cassettes for lycopene synthesis could be successfully integrated into a single locus at one time with a success rate of 57%. A 27-kb-long DNA fragment could also be precisely knocked out with a 50% positive rate in K. phaffii by introducing two DSBs simultaneously. Finally, to explore the feasibility of rapidly balancing the expression intensity of multiple genes in a metabolic pathway, a yeast combinatorial library was successfully constructed in K. phaffii using lycopene as an indicator, and an optimal combination of the metabolic pathway was identified by screening, with a yield titer of up to 182.73 mg/L in shake flask fermentation. KEY POINTS: • Rapid marker recycling based on the visualization of a green fluorescent protein • One-step multifragment integration and large fragment knockout in the genome • A random assembly of multiple DNA elements to create yeast libraries in K. phaffii.

Lactiplantibacillus plantarum X7022 Plays Roles on Aging Mice with Memory Impairment Induced by D-Galactose Through Restoring Neuronal Damage, Relieving Inflammation and Oxidative Stress.

In this study, Lactiplantibacillus plantarum X7022 was applied to ameliorate memory impairment of aging mice induced by D-galactose. The strain showed specific choloylglycine hydrolysis ability based on in vitro investigation. Morris water maze test showed L. plantarum X7022 administration improved learning ability and spatial memory of aging mice. The gavage of L. plantarum X7022 displayed a promising ability of relieving cerebral oxidative stress and hippocampal inflammatory condition according to the increased GSH level and SOD activity and decreased MDA level, as well as decreased TNF-α, IL-1ß, and IL-6 levels. The intervention with the strain could protect neuron by regulating cell apoptosis and AChE overexpression and inhibiting amyloid-ß deposition, as well as affect neuron functions by regulating CREB-BDNF signaling pathways and iNOS expression. Besides, the strain could improve fecal SCFA contents and increase the abundance of anti-inflammatory and antioxidant-related genera such as Lactobacillus, Akkermansia, and Adlercreutzia. These results suggest that L. plantarum X7022 could be a prospective therapeutic alternative for the improvement of memory impairment among the elderly.

Identification and evolution of non-traditional nitrilase from Spirosoma linguale DSM 74 with high hydration activity.

Hydration, a secondary activity mediated by nitrilase, is a promising new pathway for amide production. However, low hydration activity of nitrilase or trade-off between hydration and catalytic activity hinders its application in the production of amides. Here, natural C-terminal-truncated wild-type nitrilase, mined from a public database, obtained a high-hydration activity nitrilase as a novel evolutionary starting point for further protein engineering. The nitrilase Nit-74 from Spirosoma linguale DSM 74 was successfully obtained and exhibited the highest hydration activity level, performing 50.7 % nicotinamide formation and 87.6 % conversion to 2 mM substrate 3-cyanopyridine. Steric hindrance of the catalytic activity center and the N-terminus of the catalytic cysteine residue helped us identify three key residues: I166, W168, and T191. Saturation mutations resulted in three single mutants that further improved the hydration activity of N-heterocyclic nitriles. Among them, the mutant T191S performed 72.7 % nicotinamide formation, which was much higher than the previously reported highest level of 18.7 %. Additionally, mutants I166N and W168Y exhibited a 97.5 % 2-picolinamide ratio and 97.7 % isonicotinamide ratio without any loss of catalytic activity, which did not indicate a trade-off effect. Our results expand the screening and evolution library of promiscuous nitrilases with high hydration activity for amide formation.

Enhanced Thermostability of Geobacillus stearothermophilus α-Amylase by Rational Design of Disulfide Bond and Application in Corn Starch Liquefaction and Bread Quality Improvement.

α-Amylase (EC 3.2.1.1) from Geobacillus stearothermophilus (generally recognized as safe) exhibited thermal inactivation, hampering its further application in starch-based industries. To address this, we performed structural analyses based on molecular dynamics targeting the flexible regions of α-amylase. Subsequently, we rationally designed a thermostable mutant, AmyS1, by introducing disulfide bonds to stabilize the flexible regions. AmyS1 showed excellent thermostability without any stability-activity trade-off, giving a 40-fold longer T1/2 (1359 min) at 90 °C. Thermostability mechanism analysis revealed that the introduction of disulfide bonds in AmyS1 refined weak spots and reconfigured the protein's force network. Moreover, AmyS1 exhibited improved pH compatibility and enhanced corn starch liquefaction at 100 °C with a 5.1-fold increased product concentration. Baking tests confirmed that AmyS1 enhanced bread quality and extended the shelf life. Therefore, mutant AmyS1 is a robust candidate for the starch-based industry.

Construction of RNA silencing system of Penicillium brevicompactum and genetic manipulation of the regulator pbpcz in mycophenolic acid production.

Penicillium brevicompactum is a critical industrial strain for the production of mycophenolic acid (MPA). However, the genetic background of Penicillium brevicompactum is unclear, and there are few tools available for genetic manipulation. To investigate its gene function, we first verified the feasibility of a pair of citrate synthase promoter (Pcit) and terminator (Tcit) from P. brevicompactum by constructing a fluorescent expression cassette. Based on this, an RNAi vector was designed and constructed with reverse promoters. This study focused on the functional investigation of the pbpcz gene in P. brevicompactum, a regulator belonging to the Zn(II)2Cys6 family. RNAi was used to silence the pbpcz gene, providing a valuable tool for genetic studies in P. brevicompactum. After seven days, we observed differences in the number of spores between different phenotypes strains of pbpcz gene. Compared to the wild-type strain (WT), the spore yield of the pbpcz gene silencing mutant (M2) was only 51.4 %, while that of the pbpcz gene overexpressed mutant (SE4) was increased by 50 %. Expression levels of the three genes (brlA, abaA, and wetA) comprising conidia's central regulatory pathway were significantly reduced in the pbpcz gene silencing mutant, while fluorescence localization showed that PbPCZ protein was mainly distributed in spores. The results indicated that the pbpcz gene is critical for conidia and asexual development of P. brevicompactum. In addition, overexpressing the pbpcz gene resulted in a 30.3 % increase in MPA production compared to the wild type, with a final yield of 3.57 g/L. These results provide evidence that PbPCZ acts as a positive regulator in P. brevicompactum, controlling MPA production and regulating conidia and asexual development.

Identification, Characterization, and Computer-Aided Rational Design of a Novel Thermophilic Esterase from Geobacillus subterraneus, and Application in the Synthesis of Cinnamyl Acetate.

Investigation of a novel thermophilic esterase gene from Geobacillus subterraneus DSMZ 13552 indicated a high amino acid sequence similarity of 25.9% to a reported esterase from Geobacillus sp. A strategy that integrated computer-aided rational design tools was developed to select mutation sites. Six mutants were selected from four criteria based on the simulated saturation mutation (including 19 amino acid residues) results. Of these, the mutants Q78Y and G119A were found to retain 87% and 27% activity after incubation at 70 °C for 20 min, compared with the 19% activity for the wild type. Subsequently, a double-point mutant (Q78Y/G119A) was obtained and identified with optimal temperature increase from 65 to 70 °C and a 41.51% decrease in Km. The obtained T1/2 values of 42.2 min (70 °C) and 16.9 min (75 °C) for Q78Y/G119A showed increases of 340% and 412% compared with that in the wild type. Q78Y/G119A was then employed as a biocatalyst to synthesize cinnamyl acetate, for which the conversion rate reached 99.40% with 0.3 M cinnamyl alcohol at 60 °C. The results validated the enhanced enzymatic properties of the mutant and indicated better prospects for industrial application as compared to that in the wild type. This study reported a method by which an enzyme could evolve to achieve enhanced thermostability, thereby increasing its potential for industrial applications, which could also be expanded to other esterases.

Unravelling and engineering an operon involved in the side-chain degradation of sterols in Mycolicibacterium neoaurum for the production of steroid synthons.

BACKGROUND: Harnessing engineered Mycolicibacteria to convert cheap phytosterols into valuable steroid synthons is a basic way in the industry for the production of steroid hormones. Thus, C-19 and C-22 steroids are the two main types of commercial synthons and the products of C17 side chain degradation of phytosterols. During the conversion process of sterols, C-19 and C-22 steroids are often produced together, although one may be the main product and the other a minor byproduct. This is a major drawback of the engineered Mycolicibacteria for industrial application, which could be attributed to the co-existence of androstene-4-ene-3,17-dione (AD) and 22-hydroxy-23,24-bisnorchol-4-ene-3-one (HBC) sub-pathways in the degradation of the sterol C17 side chain. Since the key mechanism underlying the HBC sub-pathway has not yet been clarified, the above shortcoming has not been resolved so far. RESULTS: The key gene involved in the putative HBC sub-pathway was excavated from the genome of M. neoaurum by comparative genomic analysis. Interestingly, an aldolase- encoding gene, atf1, was identified to be responsible for the first reaction of the HBC sub-pathway, and it exists as a conserved operon along with a DUF35-type gene chsH4, a reductase gene chsE6, and a transcriptional regulation gene kstR3 in the genome. Subsequently, atf1 and chsH4 were identified as the key genes involved in the HBC sub-pathway. Therefore, an updated strategy was proposed to develop engineered C-19 or C-22 steroid-producing strains by simultaneously modifying the AD and HBC sub-pathways. Taking the development of 4-HBC and 9-OHAD-producing strains as examples, the improved 4-HBC-producing strain achieved a 20.7 g/L production titer with a 92.5% molar yield and a 56.4% reduction in byproducts, and the improved 9-OHAD producing strain achieved a 19.87 g/L production titer with a 94.6% molar yield and a 43.7% reduction in byproduct production. CONCLUSIONS: The excellent performances of these strains demonstrated that the primary operon involved in the HBC sub-pathway improves the industrial strains in the conversion of phytosterols to steroid synthons.

Engineering the metabolism and morphology of the filamentous fungus Trichoderma reesei for efficient L-malic acid production.

L-malic acid (MA) is a vital platform chemical with huge market demand because of its broad industrial applications. A cell factory for MA production was engineered by strengthening the intrinsic pathway without inserting foreign genes into Trichoderma reesei. The native MA transporter gene in the T. reesei genome was characterized (trmae1), and its overexpression significantly improved MA production, which increased from 2 to 56.24 g/L. Native pyruvate carboxylase, malate dehydrogenase, malic enzyme, and glucose transporter were overexpressed further to improve the titer and yield of MA production. Fungal morphology was adapted to produce MA in the fermenter by deleting gul1. A titer of 235.8 g/L MA was produced from the final engineered strain in a 5-L fermenter with a yield of 1.48 mol of MA per mol of glucose and productivity of 1.23 g/L/h. This study provides novel insights for understanding and remodeling the MA synthesis pathway.

Improved cryptic plasmids in probiotic Escherichia coli Nissle 1917 for antibiotic-free pathway engineering.

The engineered probiotic Escherichia coli Nissle 1917 (EcN) is expected to be employed in the diagnosis and treatment of various diseases. However, the introduced plasmids typically require antibiotics to maintain genetic stability, and the cryptic plasmids in EcN are usually eliminated to avoid plasmid incompatibility which may change the inherent probiotic characteristics. Here, we provided a simple design to minimize the genetic change of probiotics by eliminating native plasmids and reintroducing the recombinants carrying functional genes. Specific insertion sites in the vectors showed significant differences in the expression of fluorescence proteins. Selected integration sites were applied in the de novo synthesis of salicylic acid, leading to a titer of 142.0 ± 6.0 mg/L in a shake flask with good production stability. Additionally, the design successfully realized the biosynthesis of ergothioneine (45 mg/L) by one-step construction. This work expands the application scope of native cryptic plasmids to the easy construction of functional pathways. KEY POINTS: • Cryptic plasmids of EcN were designed to express exogenous genes • Insertion sites with different expression intensities in cryptic plasmids were provided • Target products were stably produced by engineering cryptic plasmids.

Simple and cheap CRISPR/Cas12a biosensor based on plug-and-play of DNA aptamers for the detection of endocrine-disrupting compounds.

Endocrine-disrupting compounds (EDCs) are widely distributed in the environment. Here, we present a CRISPR/Cas12a (CAS) biosensor based on DNA aptamers for point-of-care detection of EDCs. Two typical EDCs, 17ß-estradiol (E2) and bisphenol A (BPA), were selected to be detected by the CAS biosensors via the plug-and-play of their DNA aptamers. The results indicated that the performance of the CAS biosensors can be well regulated by controlling the trans-cleavage activity of Cas12a on a single-stranded DNA reporter and optimizing the sequence and ratio of DNA aptamer and activator DNA. Ultimately, two reliable and specific biosensors were developed, with the linear range and limit of detection of 0.2-25 nM and 0.08 nM for E2 and of 0.1-250 nM and 0.06 nM for BPA, respectively. Compared to the existing detection methods, the CAS biosensors showed higher reliability and sensitivity with simple operation, short detection time, and no costly equipment.

Driving the conversion of phytosterol to 9α-hydroxy-4-androstene-3,17-dione in Mycolicibacterium neoaurum by engineering the supply and regeneration of flavin adenine dinucleotide.

BACKGROUND: The conversion of phytosterols to steroid synthons by engineered Mycolicibacteria comprises one of the core steps in the commercial production of steroid hormones. This is a complex oxidative catabolic process, and taking the production of androstenones as example, it requires about 10 equivalent flavin adenine dinucleotide (FAD). As the high demand for FAD, the insufficient supply of FAD may be a common issue limiting the conversion process. RESULTS: We substantiated, using the production of 9α-hydroxy-4-androstene-3,17-dione (9-OHAD) as a model, that increasing intracellular FAD supply could effectively increase the conversion of phytosterols into 9-OHAD. Overexpressing ribB and ribC, two key genes involving in FAD synthesis, could significantly enhance the amount of intracellular FAD by 167.4% and the production of 9-OHAD by 25.6%. Subsequently, styrene monooxygenase NfStyA2B from Nocardia farcinica was employed to promote the cyclic regeneration of FAD by coupling the oxidation of nicotinamide adenine dinucleotide (NADH) to NAD+, and the production of 9-OHAD was further enhanced by 9.4%. However, the viable cell numbers decreased by 20.1%, which was attributed to sharply increased levels of H2O2 because of the regeneration of FAD from FADH2. Thus, we tried to resolve the conflict between FAD regeneration and cell growth by the overexpression of catalase and promotor replacement. Finally, a robust strain NF-P2 was obtained, which could produce 9.02 g/L 9-OHAD after adding 15 g/L phytosterols with productivity of 0.075 g/(L h), which was 66.7% higher than that produced by the original strain. CONCLUSIONS: This study highlighted that the cofactor engineering, including the supply and recycling of FAD and NAD+ in Mycolicibacterium, should be adopted as a parallel strategy with pathway engineering to improve the productivity of the industrial strains in the conversion of phytosterols into steroid synthons.

Boosting the epoxidation of squalene to produce triterpenoids in Saccharomyces cerevisiae.

BACKGROUND: Polycyclic triterpenoids (PTs) are common in plants, and have attracted considerable interest due to their remarkable biological activities. Currently, engineering the ergosterol synthesis pathway in Saccharomyces cerevisiae is a safe and cost-competitive way to produce triterpenoids. However, the strict regulation of ERG1 involved in the epoxidation of squalene limits the triterpenoid production. RESULTS: In this study, we found that the decrease in ERG7 protein level could dramatically boost the epoxidation of squalene by improving the protein stability of ERG1. We next explored the potential factors that affected the degradation process of ERG1 and confirmed that ERG7 was involved in the degradation process of ERG1. Subsequently, expression of four different triterpene cyclases utilizing either 2,3-oxidosqualene or 2,3:22,23-dioxidosqualene as the substrate in ERG7-degraded strains showed that the degradation of ERG7 to prompt the epoxidation of squalene could significantly increase triterpenoid production. To better display the potential of the strategy, we increased the supply of 2,3-oxidosqualene, optimized flux distribution between ergosterol synthesis pathway and ß-amyrin synthesis pathway, and modified the GAL-regulation system to separate the growth stage from the production stage. The best-performing strain ultimately produced 4216.6 ± 68.4 mg/L of ß-amyrin in a two-stage fed-fermentation (a 47-fold improvement over the initial strain). CONCLUSIONS: This study showed that deregulation of the native restriction in ergosterol pathway was an effective strategy to increase triterpenoid production in yeast, which provided a new insight into triterpenoids biosynthesis.

The identification of a robust leucine dehydrogenase from a directed soil metagenome for efficient synthesis of L-2-aminobutyric acid.

L-2-aminobutyric acid (L-2-ABA) is a chiral precursor for the synthesis of anti-epileptic drug levetiracetam and anti-tuberculosis drug ethambutol. Asymmetric synthesis of L-2-ABA by leucine dehydrogenases has been widely developed. However, the limitations of natural enzymes, such as poor stability, low catalytic efficiency, and inhibition of high-concentration substrates, limit large-scale applications. Herein, by directed screening of a metagenomic library from unnatural amino acid-enriched environments, a robust leucine dehydrogenase, TvLeuDH, was identified, which exhibited high substrate tolerance and excellent enzymatic activity towards 2-oxobutyric acid. In addition, TvLeuDH has strong affinity for NADH. Subsequently, a three-enzyme co-expression system containing L-threonine deaminase, TvLeuDH, and glucose dehydrogenase was established. By optimizing reaction conditions, 1.5 M L-threonine could be converted to L-2-ABA with a 99% molar conversion rate and a space-time yield of 51.5 g·L-1 ·h-1 . In this process, no external coenzyme was added. The robustness of TvLeuDH allowed the reaction to be performed without the addition of extra salt as the buffer, demonstrating the simplest reaction system currently reported. These unique properties for the efficient and environmentally friendly production of chiral amino acids make TvLeuDH a particularly promising candidate for industrial applications, which reveals the great potential of directed metagenomics for industrial biotechnology.

The Ras small GTPase RSR1 regulates cellulase production in Trichoderma reesei.

BACKGROUND: Lignocellulose is the most abundant renewable resource in the world and has attracted widespread attention. It can be hydrolyzed into sugars with the help of cellulases and hemicellulases that are secreted by filamentous fungi. Several studies have revealed that the Ras small GTPase superfamily regulates important cellular physiological processes, including synthesis of metabolites, sporulation, and cell growth and differentiation. However, it remains unknown how and to what extent Ras small GTPases participate in cellulase production. RESULTS: In this study, we found that the putative Ras small GTPase RSR1 negatively regulated the expression of cellulases and xylanases. Deletion of rsr1 (∆rsr1) significantly increased cellulase production and decreased the expression levels of ACY1-cAMP-protein kinase A (PKA) signaling pathway genes and the concentration of intracellular cyclic adenosine monophosphate (cAMP). Loss of acy1 based on ∆rsr1 (∆rsr1∆acy1) could further increase cellulase production and the expression levels of cellulase genes, while overexpression of acy1 based on ∆rsr1 (∆rsr1-OEacy1) significantly reduced cellulase production and transcriptional levels of cellulase genes. In addition, our results revealed that RSR1 negatively controlled cellulase production via the ACY1-cAMP-PKA pathway. Transcriptome analysis revealed significantly increased expression of three G-protein coupled receptors (GPCRs; tre62462, tre58767, and tre53238) and approximately two-fold higher expression of ACE3 and XYR1, which transcriptionally activated cellulases with the loss of rsr1. ∆rsr1∆ tre62462 exhibited a decrease in cellulase activity compared to ∆rsr1, while that of ∆rsr1∆tre58767 and ∆rsr1∆tre53238 showed a remarkable improvement compared to ∆rsr1. These findings revealed that GPCRs on the membrane may sense extracellular signals and transmit them to rsr1 and then to ACY1-cAMP-PKA, thereby negatively controlling the expression of the cellulase activators ACE3 and XYR1. These data indicate the crucial role of Ras small GTPases in regulating cellulase gene expression. CONCLUSIONS: Here, we demonstrate that some GPCRs and Ras small GTPases play key roles in the regulation of cellulase genes in Trichoderma reesei. Understanding the roles of these components in the regulation of cellulase gene transcription and the signaling processes in T. reesei can lay the groundwork for understanding and transforming other filamentous fungi.

Simultaneous Production of Cellulase and ß-Carotene in the Filamentous Fungus Trichoderma reesei.

ß-Carotene is an indispensable additive in beverage, cosmetic, feed, and pharmaceutical production. The fermentation industry annually generates abundant waste mycelia from Trichoderma reesei (T. reesei), a pivotal industrial strain for cellulase and heterologous protein production. In this study, we constructed a T. reesei cell factory for ß-carotene production for the first time. Four key enzymes, CarRP, CarB, GGS1/CrtE, and HMG1, were overexpressed in T. reesei. The concentrations of medium components, including tryptone and glucose, were optimized. The modified strain accumulated ß-carotene at a titer of 218.8 mg/L in flask culture. We achieved cellulase production (FPase, 22.33 IU/mL) with the concomitant production of ß-carotene (286.63 mg/L) from T. reesei in a jar. Overall, this study offers a novel and unique approach to address the costly waste mycelium management process using T. reesei industrial strains that simultaneously produce proteins and carotenoids.

Mechanism of Zn2+ regulation of cellulase production in Trichoderma reesei Rut-C30.

BACKGROUND: Trichoderma reesei Rut-C30 is a hypercellulolytic mutant strain that degrades abundant sources of lignocellulosic plant biomass, yielding renewable biofuels. Although Zn2+ is an activator of enzymes in almost all organisms, its effects on cellulase activity in T. reesei have yet to be reported. RESULTS: Although high concentrations of Zn2+ severely suppressed the extension of T. reesei mycelia, the application of 1-4 mM Zn2+ enhanced cellulase and xylanase production in the high-yielding cellulase-producing Rut-C30 strain of T. reesei. Expression of the major cellulase, xylanase, and two essential transcription activator genes (xyr1 and ace3) increased in response to Zn2+ stimulation. Transcriptome analysis revealed that the mRNA levels of plc-e encoding phospholipase C, which is involved in the calcium signaling pathway, were enhanced by Zn2+ application. The disruption of plc-e abolished the cellulase-positive influence of Zn2+ in the early phase of induction, indicating that plc-e is involved in Zn2+-induced cellulase production. Furthermore, treatment with LaCl3 (a plasma membrane Ca2+ channel blocker) and deletion of crz1 (calcineurin-responsive zinc finger transcription factor 1) indicated that calcium signaling is partially involved in this process. Moreover, we identified the zinc-responsive transcription factor zafA, the transcriptional levels of which declined in response to Zn2+ stress. Deletion of zafA indicates that this factor plays a prominent role in mediating the Zn2+-induced excessive production of cellulase. CONCLUSIONS: For the first time, we have demonstrated that Zn2+ is toxic to T. reesei, although promotes a marked increase in cellulase production. This positive influence of Zn2+ is facilitated by the plc-e gene and zafA transcription factor. These findings provide insights into the role of Zn2+ in T. reesei and the mechanisms underlying signal transduction in cellulase synthesis.