Igor G. Sinelnikov Head of the Laboratory Ph.D. (Chemistry)
	+7 (499) 135-12-29 ext. 797 sinelnikov.i@list.ru

Keywords
Aspergillus niger, Trichoderma reesei, recombinant enzymes, food-grade enzymes, CRISPR/Cas9 genome editing, random mutagenesis, high-throughput screening

Research Areas

The laboratory develops microbial expression platforms for the production of recombinant enzymes for food and industrial applications. Research is organized around two interconnected directions.

• Direction 1. Development of mutant microbial strains with improved productivity

The central objective is to construct platform strains of Aspergillus niger and Trichoderma reesei with an optimized secretory background for the production of secreted recombinant proteins. The resulting expression system serves as a universal chassis suitable for expression of diverse target genes without re-engineering the host strain.

Optimization of the secretory background involves sequential deletion of genes encoding extracellular proteases and dominant secretome components. This reduces proteolytic degradation of the target protein and frees up the capacity of the cellular secretory machinery. Synthetic integration sites are introduced into the vacated genomic loci, enabling targeted multicopy integration of expression cassettes into pre-characterized regions of the genome.

To support this approach, the laboratory has developed a proprietary CRISPR/Cas9-based genome editing toolkit that operates in both model organisms. In parallel, research is underway on morphology engineering of filamentous fungi — controlling hyphal growth form in submerged culture to improve mass transfer and increase yields of secreted proteins.

• Direction 2. Construction of industrial enzyme-producing strains and downstream processing

Building on the platform strains developed under Direction 1, the laboratory constructs producers of specific target enzymes selected in view of domestic market needs and import substitution priorities. Work includes design of expression cassettes, optimization of cultivation conditions, and development of enzyme purification protocols. In the longer term, this direction will encompass scale-up of fermentation processes from laboratory to pilot scale, deposition of strains in national culture collections, and preparation of regulatory documentation for enzyme product registration.

Basic Research Methods

• Genome editing toolkit.
  A unified CRISPR/Cas9 system for introducing targeted knockouts has been established. The system operates in both A. niger and T. reesei using a single selectable marker. A Golden Gate-based protocol for guide RNA replacement enables iterative genome modifications with marker recycling.
• High-throughput screening. Microplate-based assays for catalytic activity; mutagenesis with screening of 500–1000 clones per round.
• Classical methods of biochemistry and genetic engineering.

Most Significant Results

• A CRISPR/Cas9-based genome editing system has been developed for the filamentous fungi A. niger and T. reesei, enabling targeted knockouts, deletions, and insertions. The system supports iterative modifications through a recyclable marker strategy, avoiding integration of undesirable sequences into the production strain genome.
• A library of A. niger mutant strains with improved yields of natively secreted proteins has been obtained through optimization of the secretory background — deletion of extracellular protease genes.
• A high-throughput screening system for mutant strains and enzyme variants has been established in microplate format using a robotic platform (Tecan EVO150). The system is capable of analyzing libraries of several hundred clones per screening round and is applicable to both rationally designed variants and libraries generated by chemical mutagenesis.