Transfer of disulfide bond formation modules via yeast artificial chromosomes promotes the expression of heterologous proteins in Kluyveromyces marxianus.

Kluyveromyces marxianus is a food-safe yeast with great potential for producing heterologous proteins. Improving the yield in K. marxianus remains a challenge and incorporating large-scale functional modules poses a technical obstacle in engineering. To address these issues, linear and circular yeast artificial chromosomes of K. marxianus (KmYACs) were constructed and loaded with disulfide bond formation modules from Pichia pastoris or K. marxianus. These modules contained up to seven genes with a maximum size of 15 kb. KmYACs carried telomeres either from K. marxianus or Tetrahymena. KmYACs were transferred successfully into K. marxianus and stably propagated without affecting the normal growth of the host, regardless of the type of telomeres and configurations of KmYACs. KmYACs increased the overall expression levels of disulfide bond formation genes and significantly enhanced the yield of various heterologous proteins. In high-density fermentation, the use of KmYACs resulted in a glucoamylase yield of 16.8 g/l, the highest reported level to date in K. marxianus. Transcriptomic and metabolomic analysis of cells containing KmYACs suggested increased flavin adenine dinucleotide biosynthesis, enhanced flux entering the tricarboxylic acid cycle, and a preferred demand for lysine and arginine as features of cells overexpressing heterologous proteins. Consistently, supplementing lysine or arginine further improved the yield. Therefore, KmYAC provides a powerful platform for manipulating large modules with enormous potential for industrial applications and fundamental research. Transferring the disulfide bond formation module via YACs proves to be an efficient strategy for improving the yield of heterologous proteins, and this strategy may be applied to optimize other microbial cell factories.

Coupling thermotolerance and high production of recombinant protein by CYR1N1546K mutation via cAMP signaling cascades.

In recombinant protein-producing yeast strains, cells experience high production-related stresses similar to high temperatures. It is possible to increase recombinant protein production by enhancing thermotolerance, but few studies have focused on this topic. Here we aim to identify cellular regulators that can simultaneously activate thermotolerance and high yield of recombinant protein. Through screening at 46 °C, a heat-resistant Kluyveromyces marxianus (K. marxianus) strain FDHY23 is isolated. It also exhibits enhanced recombinant protein productivity at both 30 °C and high temperatures. The CYR1N1546K mutation is identified as responsible for FDHY23's improved phenotype, characterized by weakened adenylate cyclase activity and reduced cAMP production. Introducing this mutation into the wild-type strain greatly enhances both thermotolerance and recombinant protein yields. RNA-seq analysis reveals that under high temperature and recombinant protein production conditions, CYR1 mutation-induced reduction in cAMP levels can stimulate cells to improve its energy supply system and optimize material synthesis, meanwhile enhance stress resistance, based on the altered cAMP signaling cascades. Our study provides CYR1 mutation as a novel target to overcome the bottleneck in achieving high production of recombinant proteins under high temperature conditions, and also offers a convenient approach for high-throughput screening of recombinant proteins with high yields.

Deep Generative Mixture Model for Robust Imbalance Classification.

Discovering hidden pattern from imbalanced data is a critical issue in various real-world applications. Existing classification methods usually suffer from the limitation of data especially for minority classes, and result in unstable prediction and low performance. In this paper, a deep generative classifier is proposed to mitigate this issue via both model perturbation and data perturbation. Specially, the proposed generative classifier is derived from a deep latent variable model where two variables are involved. One variable is to capture the essential information of the original data, denoted as latent codes, which are represented by a probability distribution rather than a single fixed value. The learnt distribution aims to enforce the uncertainty of model and implement model perturbation, thus, lead to stable predictions. The other variable is a prior to latent codes so that the codes are restricted to lie on components in Gaussian Mixture Model. As a confounder affecting generative processes of data (feature/label), the latent variables are supposed to capture the discriminative latent distribution and implement data perturbation. Extensive experiments have been conducted on widely-used real imbalanced image datasets. Experimental results demonstrate the superiority of our proposed model by comparing with popular imbalanced classification baselines on imbalance classification task.

Research on Integrated Innovation Design Education for Cultivating the Innovative and Entrepreneurial Ability of Industrial Design Professionals.

In the industrial design industry, innovation and entrepreneurship made by professional and technical talents will become an important trend in the future development of the economy and society. The great changes in the design industry, such as the diversification and complication of design objects, the relatively restricted knowledge structure of design talents, the lack of entrepreneurial experience, and other reasons, have led to the low success rate of industrial design talent entrepreneurship. The purpose of this research is to analyze and explore the effects of integrated innovation design teaching modes and methods of cultivating the innovative and entrepreneurial abilities of industrial design professionals to help industrial design professionals to improve their success rate of establishing new companies. This research also aims to improve the innovative and entrepreneurial ability of industrial design professionals in terms of integrated innovation and design teaching reform by integrating the theories and methods of industrial design professional training links such as the objectives, teaching contents, teaching modes and methods, performance evaluation of innovation, and design education, combining these with the requirements of the innovative and entrepreneurial ability training of industrial design professionals. In this study, the educational reform of integrated innovation design in a specific Chinese university is taken as an example to carry out theoretical research and practical application. The results of this research show that the theory and method of integrated innovation of design education have made significant contributions to the enhancement of the innovative and entrepreneurial ability of industrial design professionals. This research also reveals a feasible direction for constructing an industrial design innovative and entrepreneurial talent training system that adapts to the transformation and upgrading of the design industry. Meanwhile, methods and teaching modes applied in this research can be promoted in relevant or similar majors to make innovation and entrepreneurship education in colleges and universities more professional, stimulate students' entrepreneurial consciousness, and improve students' abilities of innovation and entrepreneurship.

Protoplast transformation of Kluyveromyces marxianus.

The dairy yeast Kluyveromyces marxianus is a promising cell factory for producing bioethanol and heterologous proteins, as well as a robust synthetic biology platform host, due to its safe status and beneficial traits, including fast growth and thermotolerance. However, the lack of high-efficiency transformation methods hampers the fundamental research and industrial application of this yeast. Protoplast transformation is one of the most commonly used fungal transformation methods, but it yet remains unexplored in K. marxianus. Here, we established the protoplast transformation method of K. marxianus for the first time. A series of parameters on the transformation efficiency were optimized: cells were collected in the late-log phase and treated with zymolyase for protoplasting; the transformation was performed at 0 °C with carrier DNA, CaCl2 , and PEG; after transformation, protoplasts were recovered in a solid regeneration medium containing 3-4% agar and 0.8 m sorbitol. By using the optimized method, plasmids of 10, 24, and 58 kb were successfully transformed into K. marxianus. The highest efficiency reached 1.8 × 104 transformants per µg DNA, which is 18-fold higher than the lithium acetate method. This protoplast transformation method will promote the genetic engineering of K. marxianus that requires high-efficiency transformation or the introduction of large DNA fragments.

Meiosis-Based Laboratory Evolution of the Thermal Tolerance in Kluyveromyces marxianus.

Kluyveromyces marxianus is the fastest-growing eukaryote and a promising host for producing bioethanol and heterologous proteins. To perform a laboratory evolution of thermal tolerance in K. marxianus, diploid, triploid and tetraploid strains were constructed, respectively. Considering the genetic diversity caused by genetic recombination in meiosis, we established an iterative cycle of "diploid/polyploid - meiosis - selection of spores at high temperature" to screen thermotolerant strains. Results showed that the evolution of thermal tolerance in diploid strain was more efficient than that in triploid and tetraploid strains. The thermal tolerance of the progenies of diploid and triploid strains after a two-round screen was significantly improved than that after a one-round screen, while the thermal tolerance of the progenies after the one-round screen was better than that of the initial strain. After a two-round screen, the maximum tolerable temperature of Dip2-8, a progeny of diploid strain, was 3°C higher than that of the original strain. Whole-genome sequencing revealed nonsense mutations of PSR1 and PDE2 in the thermotolerant progenies. Deletion of either PSR1 or PDE2 in the original strain improved thermotolerance and two deletions displayed additive effects, suggesting PSR1 and PDE2 negatively regulated the thermotolerance of K. marxianus in parallel pathways. Therefore, the iterative cycle of "meiosis - spore screening" developed in this study provides an efficient way to perform the laboratory evolution of heat resistance in yeast.