Production Hosts and Intelligent Tools
ID: 3936

Abstract: Kristala Jones Prather

The growing interest in a “biomass-based” economy has led to new efforts to construct and improve microorganisms capable of producing chemicals. The current focus is largely on liquid biofuels; however, a successful “biorefinery” is likely to be a mixed-product facility, with many compounds produced from one or more biomass-derived feeds. Identifying methods for the production of both novel biofuels and “value-added” compounds is both a challenge and an opportunity. The potential for biological conversion of feedstocks to bulk chemicals is enhanced by the availability of tools and techniques from the established discipline of Metabolic Engineering, which has enjoyed tremendous successes in the development of highly productive microorganisms for a variety of products of interest. We can also gain insights from Biocatalysis, where the choice of enzymes to mediate biotransformation of chemical substrates is based largely on consideration of the required functional group conversion without being limited by prior evidence of transformation of the full structure. The focus of Synthetic Biology on the application of engineering principles to the design and re-design of biological systems to perform prescribed tasks, and to do in a predictive, robust, and more reliable manner, has led to the development of new tools and methodologies for both design and assembly that further enhance our ability to engineer microbial chemical factories. Our group is interested in applying principles from each of these intellectual arenas towards the design and construction of novel biosynthetic pathways for specified target compounds. In particular, we will present our work on the microbial synthesis of glucaric acid (a dicarboxylic acid) and hydroxyvaleric acids as value-added compounds from biomass. We will also discuss the challenges of constructing productive organisms in a predictive manner through the use of well-characterized biological parts.













Xue-Ming Zhao

Genome shuffling for industrial biotechnology: Chinese practices

Xue-Ming ZHAO

Department of Biochemical Engineering

School of Chemical Engineering & Technology

Key Laboratory of Systems Bioengineering, Ministry of Education

Tianjin University, Tianjin 300072, China

Email: xmzhao@tju.edu.cn



Metabolic engineering emerged, just over 18 years ago, as the discipline that utilizes modern genetic tools for the construction of organisms capable of fuel and chemical production. Metabolic engineering exploits an integrated, systems-level approach for optimizing a desired cellular property or phenotype; and great strides have been made within this scope and context during the past 18 years. Although this ‘rational design’ approach has been successful in many applications, it was established early on that the interconnectivity and sheer complexity of biological networks often preclude the recognition of simple genotype–phenotype relationships to guide these modifications. Recent advances in genomic technologies have improved the ability to identify the genes responsible for the desired trait. Nevertheless, only when one or few well-known genes encode the desired trait, the recombinant strain construction is relatively easy and feasible. The most desired traits, such as cell growth rate, product yield and tolerance, are quantitatively and continuously distributed phenotypes determined by cumulative contribute of multiple genes. For example, it is speculated that the genetics of the ethanol tolerance basic trait involve more than 250 genes. Such challenges led to the development of some new concepts called ‘inverse metabolic engineering’, ‘evolutionary metabolic engineering ’, and ‘combinatorial engineering’ for cell optimization. As Gregory Stephanopoulos proposed that real metabolic engineers need do ‘rational and combinatorial’ both.



Whole-genome shuffling allows for the generation of combinatorial libraries of complex progeny from a few previously selected parental strains exhibiting subtle improvements in a desired property. Thus, genome shuffling is clearly an important metabolic engineering tool for strain improvement. Since 2002, genome shuffling has been used to engineer a myriad of other complex phenotypes, there were more than 50 papers published. Among them, about 30 published papers from China. In this presentation we will illustrate those examples from China: Genome shuffing to improve thermotolerance, ethanol tolerance and ethanol productivity of Saccharomyces cerevisiae; Genome-shuffling improved acid tolerance, glucose tolerance and L-lactic acid volumetric productivity in Lactobacillus rhamnosus; Improved production of pristinamycin by Streptomyces pristinaespiralis; Rapid Improvement in Lipase Production of Penicillium expansum; Genome shuffling in the ethanologenic yeast Candida krusei to improve acetic acid tolerance; Evolution of Streptomyces pristinaespiralis for resistance and production of pristinamycin by genome shuffling; Trait improvement of riboflavin-producing Bacillus subtilis by genome shuffling; Astaxanthin-producing strain breeding by genome shuffling; Enhancement and selective production of avermectin B by recombinants of Streptomyces avermitilis; Enhancement of monacolin K production by genome shuffling between Aspergillus terreus and Monascus anka; Screening and breeding of high taxol producing fungi by genome shuffling; Whole Genome Shuffling to Enhance Activity of Fibrinolytic Enzyme producing Strains; Natamycin -producing strain breeding by genome shuffling; Neutral protease production by aspergillus sojae; Improvement of teicoplanin-producing strain by Actinoplanes teichomyceticus; Mutation and a high-throughput screening method for improving the production of Epothilones of Sorangium; High-throughput method for screening of rapamycin-producing strains of Streptomyces hygroscopicus by cultivation in 96-well microtiter plates; etc.

















Gaëtan MICHEL

This presentation will outline how a revolutionary pipeline of specialty ingredients manufactured from renewable sources, using a patented and environmentally friendly technology, was developed and implemented by KitoZyme, a Belgian company founded in 2000.



This pioneer pipeline of functional biopolymers provides cost-effective and scientifically-proven solutions for customers who develop high added-value applications in four key markets:

• Nutraceuticals

• Cosmetics

• Biomedical (medical devices and drug delivery systems)

• Beverages



For the first time ever, these biopolymers are industrially extracted and purified from renewable fungal sources from the food and biotech industries. The processes and production units were designed by KitoZyme to meet strict quality standards (ISO9001:2008, GMP, HACCP), regulation and environmental constraints, traceability, safety, flexibility and competitiveness. Recycling and recovery of co-products are also implemented.



KitoZyme has established and validated several industrial production units from non-GMO fungal sources, carefully selected to achieve optimum production cost, traceability, safety, purity and specifications in terms of purity, molecular characteristics and performances :

• The mycelium of Aspergillus niger, a microscopic fungus, a co-product of the food and pharma citric acid producing industry

• Agaricus bisporus, edible white mushroom from the food industry



The pipeline is currently composed of the following polysaccharides :

• The first ultra-pure chitosan (polyglucosamine) of non-animal origin produced according to cGMP (FDA-EMEA), a highly prized biocompatible biomaterial for medical devices and functional excipient for advanced drug delivery systems

• The first chitosan of vegetal origin with health benefits for nutraceutical applications, like weight management and cholesterol control

• The first chitosan succinamide of non-animal origin, an alternative to silicons for a silky touch in personal care and skin care products

• Chitin-glucan, a unique and patented copolymer of poly(N-acetyl-glucosamine) and beta(1,3) glucan, with health benefits for nutraceutical applications, like management of oxidative stress and transit management

• Ultra-fine chitin-glucan for cosmetic skin care, with skin rejuvenation and anti-age properties proven by three clinical studies

• The first non-animal chitosan for beverage stabilization

• Chitin-glucan for must clarification and wine fining



A whole technological platform was designed based on this family of biopolymers, relying on an extensive know-how in analytical chemistry, functionalization chemistry, processing, formulation, physico-chemical and biological properties.



Opportunities for value creation are endless, and KitoZyme is establishing long-term collaborative ventures with solid and innovative partners.















Mariet van der Werf

Industrial biotechnology is increasingly applied in the production of a large number of chemicals (i.e. bioethanol, citric acid, lysine, 1,3-propanediol). As the cost-price of large scale industrial fermentations is primarily determined by the substrate costs, increasingly cheap lignocellulosic biomass streams are used in order to make these production processes more cost-effective and, at the same time, more environmentally friendly. Currently, the selection of the microbial production host is primarily based on its potential to produce the product of interest or based on prior experience with the micro-organism.

Lignocellulosic feedstocks consists out of a mixture of different fermentable sugars (i.e. glucose, xylose, arabinose, galactose, mannose, etc). Moreover, depending on the pretreatment and hydrolysis processes applied to convert the lignocellulose into the fermentable sugars, high salt concentrations as well as inhibitors are present in these feedstock hydrolysates. A substrate oriented approach towards production host selection could therefore avoid extensive metabolic engineering as several substrate utilization routes are required and only one biosynthesis route. Moreover, feedstock hydrolysate-related growth inhibition can be minimized in this way.

We have compared the performance of six industrially relevant microorganisms i.e. two bacteria (Escherichia coli and Corynebacterium glutamicum) two yeast (Saccharomyces cerevisiae and Pichia pastoris) and two fungi (Aspergillus niger and Trichoderma reesei), for their (i) ability to utilize monosaccharides present in lignocellulosic hydrolysates, (ii) resistance against inhibitors present in lignocellulosic feedstocks, (iii) their ability to utilize and grow on different renewable feedstock hydrolysates (corn stover, wheat straw, bagasse and willow wood). The feedstock hydrolysates were generated in two manners: (i) thermal pretreatment under mild acid conditions followed by enzymatic hydrolysis and (ii) using TNO’s Biosulfurol process (non-enzymatic method in which the lignocellulose is pretreated and hydrolyzed by concentrated sulphuric acid in combination with recycling of >99% of the sulphuric acid). Moreover, their ability to utilize waste glycerol from the biodiesel industry was evaluated. Large differences in the performance of these micro-organisms were observed.











Moderator: Kristala Prather, Massachusetts Institute of Technology (United States)

Presenter 1: Rational Design of Microbial Chemical Factories: Enzymes as Interchangeable Parts
Kristala Jones Prather, MIT, (United States)  [Confirmed]

Presenter 2: Genome Shuffling for Industrial Biotechnology: Chinese Practices  
Xue-Ming Zhao, Tianjin University, (China)  [Confirmed]

Presenter 3: Ground-Breaking Technology Platform of Specialty Ingredients From Renewable Fungal Sources 
Gaëtan MICHEL, KitoZyme, (Belgium)  [Confirmed]

Presenter 4 (if necessary): Selection of Microbial Production Host for Converting Lignocellulose into Bioproducts 
Mariet van der Werf, TNO Quality of Life, (Netherlands)  [Confirmed]

Panel Organizer:
Matthew Carr, Biotechnology Industry Organization, (United States)

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