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Expert Details

Filamentous Fungal Strain Engineering

ID: 739763 California, USA

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Expert has worked in Industrial Biotechnology for over 35 years to genetically engineer various filamentous fungi for protein production. Generally, he worked as a Bench Scientist at the enzyme companies Genencor, Danisco and DuPont.

Expert has experienced startup environments and large corporations. His career has spanned classical mutagenesis and selection and he's developed screening methods to alter phenotype and create enzyme hyperexpression strains. Expert has carried out media development and DOE to optimize strain characteristics.

He has grown filamentous fungi at various scale: microtiter plate, shake flasks and bioreactors ranging from 5L glass vessel, 15L pressurized stainless steel, scaleup of manufacturing fermentors sized 8K, 12K, 50K and 250K liters. Expert developed a novel, sterile Aspergillus niger pectinase liquid feeding method which improved protein productivity, reliability and product quality. He has extensively worked with and transformed Trichoderma reesei to genetically engineer strains for expression of homologous and heterologous protein expression, both as single protein and as multiprotein expression hosts. Having molecular biology experience in fungal transformation, cloning, expression, gene deletion, vector construction, library construction, recombination and CRISPR methodologies, he's isolated filamentous fungi from the environment for culturing, genome sequencing, in-vitro and in-silico gene discovery and cloning.

Expert is an inventor on over two dozen patent families predominately in the field of fungal strain genetic engineering and enzyme application technology. The past few years, he has been providing consulting services regarding filamentous fungal protein expression systems. As sole proprietor, he has provided both remote advice and in person bench guidance to strain and fermentation engineers engaged in the engineering and cultivation of filamentous fungi, predominately T.reesei.

Expertise in:
• expression of homologous and heterologous protein in filamentous fungi
• creation of hyperexpression fungal production strains
• recombination methods in filamentous fungi
• transformation of fungi
• vector construction (yeast assembly, Gibson, Gateway, restriction cloning)
• in-vitro and in silico gene discovery and cloning
• development of strain screening methods


Year Degree Subject Institution
Year: 1985 Degree: BS Subject: Cell and Molecular Biology Institution: San Francisco State University

Work History

Years Employer Title Department
Years: 2019 to Present Employer: Undisclosed Title: Principal Department:
- Provide consultation services regarding filamentous fungal protein expression systems and fermentation.
- Ongoing development of a proprietary T.reesei protein cell factory expressing mammalian protein for a young European company in the Ag space.
- Providing hands on training, mentoring to the strain and fermentation engineers regarding the genetic engineering and cultivation of Trichoderma; remote consulting and bench work on-site.
- Offering other non-competing clients expression and fungal microbiology advice.
Years Employer Title Department
Years: 2016 to 2018 Employer: Amyris Title: Senior Scientist Department:
- Created a T. reesei strain expressing a Biogen proprietary human IgG antibody.
- Established T. reesei transformation system consisting of selection marker vectors, auxotrophic strains, CRISPR engineering vectors, adapted microtiter plate based expression and genomic extraction.
- Demonstrated use of CRISPR for gene engineering.
- Used Genotype Specification Language for design of DNA for automated assembly of transformation vectors.
- Performed pathway engineering of a small molecule product in S. cerevisiae using homing endonuclease multi-site recombination and CRISPR locus directed integration of vectors.
Years Employer Title Department
Years: 2011 to 2016 Employer: DuPont Industrial Biosciences Title: Senior Scientist Department:
- Co-inventor of patent applications applied to T. reesei. Developed a single step method for introducing DNA into T. reesei using CRISPR, without marker integration. Co-developed a method to silently barcode strains allowing unique strain identification by MS/MS from protein product broth.
- Created heterologous alpha and glucoamylase production strains in T. reesei. Performed host deletions, expressed native, codon variant and hybrid enzyme molecules in T. reesei single locus strain. Designed codon optimized genes which achieved >10X increase in protein expression of two heterologous fungal enzymes.
- Led Cellulosic Ethanol strain development as task leader for three years. Worked with a diverse team of scientists and RA’s to coordinate the creation of project strains. These strains included single gene expression, deletion strains and tuned expression of multiple genes for creation of biomass hydrolysis strains. Various individual cellulase genes, gene combinations and gene libraries were expressed for biomass hydrolytic studies. A progression of gene and gene combinations were engineered to create biomass production strains.
- Mined public and private genomes for homologous families of cellulases, hemicellulases and b-glucosidases. Genes were cloned by PCR and DNA synthesis. One project mined fungal collections to find putative novel thermotolerant fungi based on isolate habitat descriptions. Performed natural isolate collection with strain identification by ITS sequence. 20 novel thermotolerant fungal genomes were sequenced using combined Pacific Biosciences SMRT and Illumina sequencing methods. Genome sequences were mined for enzyme genes, cloned, expressed, characterized; high performing enzymes were incorporated into next generation biomass degrading strains.
- Introduced yeast DNA assembly construction methods to Palo Alto lab. This method enabled easy construction of both large plasmid vectors >20 kb and single step assembly of multiple DNA fragments (>4 fragments).
Years Employer Title Department
Years: 2005 to 2011 Employer: Danisco USA Title: Senior Scientist Department:
- Constructed and screened T. reesei heterologous cellulase expression strains expressing combinations of native cellulases, protein engineered cellulases and heterologous cellulases from fungi and bacteria.
- Generated RNAi silencing vectors in T. reesei. Created RNAi silenced cot1 strains to alter growth morphology. Made a bifunctional RNAi vector to successfully silence two transglycosylating transcripts expressing two deleterious enzymes in the background of an A. niger glucoamylase production strain.
- Adapted cre recombinase to T. reesei to allow rapid marker removal and expression vector insertion by recombinase mediated cassette exchange (RMCE) into a determined locus.
- Deleted ku70/80 non homologous end joining (NHEJ) proteins in T. reesei, increasing homologous recombination rates.
- Invented, created a T. reesei integration host for single genes or gene library expression at a designed locus.
Years Employer Title Department
Years: 2004 to 2016 Employer: Various Title: RSO Department:
[at Palo Alto California Technology Center for Genencor, Danisco and Dupont]

- Oversaw the safe use of isotopes (32P, 14C) and ensured user compliance with safety protocols and documentation. During this 12 year period, laboratory passed two 5-year site safety inspections by the California Department of Public Health (CDPH).
Years Employer Title Department
Years: 2003 to 2005 Employer: Genencor International Title: Senior Scientist Department:
- Co-developed acetolactate synthase (ALS) selectable marker from Trichoderma reesei generating a new selectable marker and granted patent.
- Genencor expression scientist on NREL (US National Renewable Energy Laboratory) cooperative agreement (CRADA) for expression of bacterial cellulases in T. reesei. Strains delivered gram/L quantities of thermostable cellulases to NREL. Developed T. reesei strains expressing fungal and bacterial cellulases.
Years Employer Title Department
Years: 1997 to 2003 Employer: Genencor International Title: Scientist Department:
- Troubleshot failing manufacturing (>200 m3 scale) fermentations of T. reesei cellulase. Investigated at the Palo Alto lab and the Jamsankoski, Finland manufacturing plant; uncovered the failing process problem and fixed it.
- Adapted biolistic methodology for transformation of T. reesei, brought technology on-site.
Years Employer Title Department
Years: 1992 to 1997 Employer: Genencor International Title: Senior Research Associate Department:
- Engineered enzyme overexpression strains in T. reesei of native endoglucanases (egl) I, II, III and created a novel EGL3 dimer molecule; characterized N-linked glycosylation of EGL3.
- Purified gram quantities of T. reesei cellulases CBH1, CBH2, EGL1,2,3 using preparatory scale chromatography.
- Cloned the T. reesei cellulase gene eglV, using 32P labelled degenerate DNA oligonucleotides.
Years Employer Title Department
Years: 1985 to 1992 Employer: Genencor Inc Title: Research Assistant, Research Associate Department:
- Performed classical mutagenesis strain selection and screening on T. reesei, H.grisea and A. niger to create hyperexpression strains. Performed media optimization, fermentation evaluation and fermentation manufacturing scaleup at manufacturing sites in Pennsylvania, Indiana, Wisconsin and Finland.
- Developed a mutation and selection method to isolate a foundational T. reesei overexpression cellulase production strain. Proved performance at 15L fermentation scale, assisted in manufacturing scaleup. Strain and derivatives were still in manufacturing use as of 2016.
- Created an A. niger overexpression pectinase mutant strain and developed a novel hydrolyzed biomass feeding method. The strain and process were sold to Gist-Brocades.

Career Accomplishments

Publications and Patents Summary
Publications: 9
Granted Patents and Applications: 30

Fields of Expertise

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