Metabolic model-guided strain design for improved succinic acid production in Yarrowia lipolytica.

Biomass feedstock can be used for the production of biofuels or biobased chemicals to reduce anthropogenic greenhouse gas (GHG) emissions. Earlier studies about the techno‐economic performance of biofuel or biobased chemical production varied in biomass feedstock, conversion process, and other techno‐economic assumptions. This made a fair comparison between different industrial processing pathways difficult. The aim of this study is to quantify uniformly the factory‐gate production costs and the GHG emission intensity of biobased ethanol, ethylene, 1,3‐propanediol (PDO), and succinic acid, and to compare them with each other and their respective fossil equivalent products. Brazilian sugarcane and eucalyptus are used as biomass feedstock in this study. A uniform approach is applied to determine the production costs and GHG emission intensity of biobased products, taking into account feedstock supply, biobased product yield, capital investment, energy, labor, maintenance, and processing inputs. Economic performance and net avoided GHG emissions of biobased chemicals depend on various uncertain factors, so this study pays particular attention to uncertainty by means of a Monte Carlo analysis. A sensitivity analysis is also performed. As there is uncertainty associated with the parameters used for biobased product yield, feedstock cost, fixed capital investment, industrial scale, and energy costs, the results are presented in ranges. The 60% confidence interval ranges of the biobased product production costs are 0.64–1.10 US$ kg−1 ethanol, 1.18–2.05 US$ kg−1 ethylene, 1.37–2.40 US$ kg−1 1,3‐PDO, and 1.91–2.57 US$ kg−1 succinic acid. The cost ranges of all biobased products partly or completely overlap with the ranges of the production costs of the fossil equivalent products. The results show that sugarcane‐based 1,3‐PDO and to a lesser extent succinic acid have the highest potential benefit. The ranges of GHG emission reduction are 1.29–2.16, 3.37–4.12, 2.54–5.91, and 0.47–5.22 CO2eq kg−1 biobased product for ethanol, ethylene, 1,3‐PDO, and succinic acid respectively. Considering the potential GHG emission reduction and profit per hectare, the pathways using sugarcane score are generally better than eucalyptus feedstock due to the high yield of sugarcane in Brazil. Overall, it was not possible to choose a clear winner, (a) because the best performing biobased product strongly depends on the chosen metric, and (b) because of the large ranges found, especially for PDO and succinic acid, independent of the chosen metric. To quantify the performance better, more data are required regarding the biobased product yield, equipment costs, and energy consumption of biobased industrial pathways, but also about the production costs and GHG emission intensity of fossil‐equivalent products. © 2019 The Authors. Biofuels, Bioproducts, and Biorefining published by Society of Chemical Industry and John Wiley & Sons, Ltd.

Chapter 3 - Fruit and vegetable wastes for biobased chemicals

Engineered non-canonical reductive TCA pathway drives high-yield succinic acid biosynthesis in Yarrowia lipolytica.

This paper proposes a framework with six dimensions that can be useful for evaluating the potential and the current stage of a bio-based platform chemical. The framework considers the technological and strategic challenges to be fulfilled by a company that intends to lead a platform based on a bio-based chemical. A platform chemical should be an intermediate molecule, with a structure able to generate a number of derivatives, that is produced at a competitive cost, capable of allowing exploitation of the scale and scope economies, and inserted within a complete innovation ecosystem that is able to create value with governance mechanisms that are capable of allowing coordination of the innovation process and facilitation of the value capture by the focal company leading the platform, in our case the producer of the platform molecule. Based on these six dimensions, three potential platform chemicals - succinic acid, butanol and farnesene - are compared and discussed. It is possible to identify important differences concerning the technological dimensions and the strategic dimensions as well. Two of the molecules - farnesene and succinic acid - adhere to most of the conditions required to structure a platform chemical. However, the innovation ecosystem is not complete and the governance mechanisms are still under development, so it is not clear if they will be capable of allowing a favorable position for value capture by the platform leader. Butanol structuring for a platform does not seem promising. The potential of the molecule is apparently not high and the strategic initiatives are in general not focused on innovation ecosystem structuring.

Lignocellulosic feedstocks are an important resource for biorefining of renewables to bio-based fuels, chemicals, and materials. Relevant feedstocks include energy crops, residues from agriculture and forestry, and agro-industrial and forest-industrial residues. The feedstocks differ with respect to their recalcitrance to bioconversion through pretreatment and enzymatic saccharification, which will produce sugars that can be further converted to advanced biofuels and other products through microbial fermentation processes. In analytical enzymatic saccharification, the susceptibility of lignocellulosic samples to pretreatment and enzymatic saccharification is assessed in analytical scale using high-throughput or semi-automated techniques. This type of analysis is particularly relevant for screening of large collections of natural or transgenic varieties of plants that are dedicated to production of biofuels or other bio-based chemicals. In combination with studies of plant physiology and cell wall chemistry, analytical enzymatic saccharification can provide information about the fundamental reasons behind lignocellulose recalcitrance as well as about the potential of collections of plants or different fractions of plants for industrial biorefining. This review is focused on techniques used by researchers for screening the susceptibility of plants to pretreatment and enzymatic saccharification, and advantages and disadvantages that are associated with different approaches.

The International Energy Agency Bioenergy Task 42 defined biorefinery as “the sustainable processing of biomass into a spectrum of bio-based products (food, feed, chemicals, materials) and energy (fuels, power, heat)”. This includes biochemical, thermochemical, chemical, and mechanical processes. Yeast and fungi are ideal agents for biochemical biorefineries because of their metabolic versatility. This chapter will focus on yeast-biorefineries which can use inexpensive agro-industrial waste to obtain two main products: bio-oils (feed and food), and the enzyme lipase, which is a high-value product that can modify the bio-oil to get more products. The so-called “unconventional yeasts” stand out for having the ability to metabolize a variety of carbon sources. Yarrowia lipolytica, Cutaneotrichosporon oleaginosus and Rhodotorula toruloides have been described as the most efficient in terms of yield of bio-oil. For instance, Y. lipolytica can consume glycerol to produce bio-oil, lipases, organic acids, polyols and single-cell protein. Its genome is known and can be modified to overproduce the target compound or to engineer metabolic routes for other products of interest such as polyols. Therefore, this yeast has been proposed as a model for yeast-biorefineries. Besides, enzymes are one of the products with more added value that can be produced in a biorefinery. Yeasts are also very good enzyme producers or even hosts for heterologous expression of enzymes. Moreover, solid fermentation is a configuration particularly suitable to use agro-wastes in a biorefinery. Indeed, fermented solids of agro-wastes may be directly employed as biocatalysts allowing the production of more cost-competitive bio-oil fuels and biobased chemicals. This strategy may use inexpensive agro-industrial wastes as solid support/substrates for microorganism growth and lipase production, avoiding expensive steps of enzyme purification and immobilization. Recombinant lipase from Thermomyces lanuginosus produced by solid-state fermentation using agro-industrial wastes and its application to obtain biodiesel is described as example.

Organic acids like succinic and citric acids are of great interest as platform organic products that play important roles as precursors for a wide range of bio-based materials. Succinic and citric acids can be successfully produced biotechnologically from renewable resources of both hydrophilic and hydrophobic nature through efficient microbiological conversion. Yarrowia lipolytica represents one of the most versatile microbial factories in terms of organic acids production, as it easily develops and produced metabolites starting from glucidic-based and/or lipid-based substrates. The purpose of this work was to investigate the ability of Y. lipolytica to adapt to hydrophilic and hydrophobic sources and to biosynthesize important platform chemicals like succinic and citric acids. The selected strain was monitored during a batch cultivation for 192 h on 100 g/L carbon source: pure glycerol as a hydrophilic source, sunflower waste cooking oil as a hydrophobic source, and crude glycerol deriving from biodiesel production as a mixture of hydrophilic and hydrophobic sources. Cellular viability, biomass accumulation, and metabolites formation in terms of succinic acid and citric acid was monitored, and the highest results were registered for cultivations performed on waste cooking oil [10.35 ± 0.29 (log 10 ) CFU/mL, 8.15 g/L cell dry weight, 3.50 ± 0.04 g/L citric acid, and 21 ± 0.16 g/L succinic acid]. The results obtained in this work outline the industrial potential of the oleaginous yeast strain of Y. lipolytica to bioconvert the lipidic residual biomass with negative environmental implications into valuable organic compounds with wide-range applicability. • Sunflower waste cooking oil is a renewable biomass for biotechnological approaches. • Y. lipolytica produces up to 21 g/L succinic acid and 3.5 g/L citric acid from SWCO. • Y. lipolytica produces up to 14 g/L succinic acid and 4.9 g/L citric acid from CG. • Y. lipolytica produces organic acids from both hydrophilic and hydrophobic sources.

Genome-scale metabolic models (GEMs) have been developed and used in guiding systems’ metabolic engineering strategies for strain design and development. This strategy has been used in fermentative production of bio-based industrial chemicals and fuels from alternative carbon sources. However, computer-aided hypotheses building using established algorithms and software platforms for biological discovery can be integrated into the pipeline for strain design strategy to create superior strains of microorganisms for targeted biosynthetic goals. Here, I described an integrated workflow strategy using GEMs for strain design and biological discovery. Specific case studies of strain design and biological discovery using Escherichia coli genome-scale model are presented and discussed. The integrated workflow presented herein, when applied carefully would help guide future design strategies for high-performance microbial strains that have existing and forthcoming genome-scale metabolic models.

Oleaginous yeast Yarrowia lipolytica is a prospective host for production of succinic acid. The interruption of tricarboxylic acid cycle through succinate dehydrogenase gene (SDH) deletion was reported to result in strains incapable of glucose utilization and this ability had to be restored by chemical mutation or long adaptive laboratory evolution. In this study, a succinate producing strain of Y. lipolytica was engineered by truncating the promoter of SDH1 gene, which resulted in 77% reduction in SDH activity but did not impair the ability of the strain to grow on glucose. The flux toward succinic acid was further improved by overexpressing the genes in the glyoxylate pathway and the oxidative TCA branch, and expressing phosphoenolpyruvate carboxykinase from Actinobacillus succinogenes. A short adaptation on glucose reduced the lag phase of the strain and increased its tolerance to high glucose concentrations. The resulting strain produced 7.8 ± 0.0 g/L succinic acid with a yield of 0.105 g/g glucose in shake flasks without pH control, while mannitol (11.8 ± 0.8 g/L) was the main by-product. Further investigations showed that mannitol accumulation was caused by low pH stress and buffering the fermentation medium eliminated mannitol formation. In a fed-batch bioreactor in mineral medium at pH 5, at which point according to Ka values of succinic acid, the major fraction of product was in acidic form rather than dissociated form, the strain produced 35.3 ± 1.5 g/L succinic acid with 0.26 ± 0.00 g/g glucose yield.

The increasing prevalence of bio-based and biodegradable plastics as an alternative to traditional plastics derived from crude oil is a noteworthy trend. Polybutylene succinate (PBS), a plastic produced from succinic acid, is among the promising materials for the future. However, the production of bio-based succinic acid through biotechnical processes in controlled environments presents challenges. This process leads to increased costs and is currently not economically competitive compared to crude oil-based succinic acid production. In addition to succinic acid, levulinic acid is another monomer produced in the same process. A novel approach to the digestion of biomass has been developed to address the issue of biotechnological production of bio-based platform chemicals. This innovative process employs microwave radiation, pressure, and temperature to convert wood residues into succinic acid and levulinic acid. Various catalyst concentrations and biomass ratios were tested in a batch process, with high-pressure liquid chromatography (HPLC) and liquid chromatography–mass spectrometry (LC/MS) analyses revealing the formation of succinic acid, levulinic acid, formic acid, and 2-oxoglutaric acid. The results demonstrate that microwaves combined with a metal salt catalyst can be used to produce platform chemicals from lignocellulosic biomass. To further advance the continuous production of PBS, a twin-screw extruder was modified and adapted after the successful results obtained from the batch processes. This setup enables additional experiments to evaluate the transferability of batch process results to continuous reactions, facilitating the scale-up and the economic viability of the overall PBS production process in the future.

This thesis represents that pineapple processing waste provides an excellent opportunity to recover and produce valuable bio-based chemicals as a part of a biorefinery product portfolio. The current work highlights the scope and breadth of opportunity including scientific challenges that need to be addressed in valorising pineapple processing wastes. A techno-economic model is further developed to assess the economic feasibility of a biorefinery based on pineapple processing wastes. Such an integrated approach in utilizing this organic waste will establish a circular economy and diversify the market of bio-based products and chemicals.

Little information is yet available on the economic viability of the production of bio-based bulk chemicals and intermediates from white biotechnology (WB). This paper details a methodology to systematically evaluate the techno-economic prospects of present and future production routes of bio-based bulk chemicals produced with WB. Current and future technology routes are evaluated for 15 products assuming prices of fermentable sugar between 70 euro/t and 400 euro/t and crude oil prices of US $25/barrel and US $50/barrel. The results are compared to current technology routes of petrochemical equivalents. For current state-of-the-art WB processes and a crude oil price of US $25/barrel, WB-based ethanol, 1,3-propanediol, polytrimethylene terephthalate and succinic acid are economically viable. Only three WB products are economically not viable for future technology: acetic acid, ethylene and PLA. Future-technology ethylene and PLA become economically viable for a higher crude oil price (US $50/barrel). Production costs plus profits of WB products decrease by 20-50% when changing from current to future technology for a crude oil price of US $25 per barrel and across all sugar prices. Technological progress in WB can thus contribute significantly to improved economic viability of WB products. A large-scale introduction of WB-based production of economically viable bulk chemicals would therefore be desirable if the environmental impacts are smaller than those of current petrochemical production routes.

Engineering of CO2 recycling and formate metabolism for succinic acid production in Yarrowia lipolytica.

An exponential increase in our understanding of genomes, proteomes, and metabolomes provides greater impetus to address critical biotechnological issues such as sustainable production of biofuels and bio-based chemicals and, in particular, the development of improved microbial biocatalysts for use in industrial biorefineries. Because these studies involve the evaluation of large numbers of genes and proteins, high-throughput integrated robotic molecular biology platforms that have the capacity to rapidly synthesize, clone, and express heterologous gene open reading frames (ORFs) in bacteria, cell-free extracts, and yeast and to screen large numbers of expressed proteins for optimized function are an important technology for improving fungal strains for industrial production of biofuels and bio-based chemicals. We describe a system of four robotic platforms required for continuous operation of this process: (1) synthesis and screening of mutagenized gene ORFs by systematically replacing codons using an amino acid scanning mutagenesis algorithm, which evaluates all codons for functionality, in a multiplexed format to produce a library of optimized ORFs; (2) one-step construction of a synthetic yeast artificial chromosome (YAC) containing the optimized ORFs in a polyprotein cassette for expression of multiple genes such as those for enzymes in metabolic pathways or for valuable peptide or protein coproducts behind an optimized promoter with custom expression fusion tags selected for desired expression levels and protein locations inside or outside the industrial strain; (3) selection of a host strain that has been subjected to mutagenesis by irradiation and/or incubation at elevated temperatures anaerobically to produce a strain capable of robust growth in the particular biomass feedstock or waste stream or agricultural product required for profitable and environmentally friendly operations at a biorefinery and transformation of this host strain with these collections of synthetic YACs; and (4) high-throughput screening of the transformed strains for desired industrial traits. This system produces an improved industrial microorganism by manipulation of the host strain, assembly of an optimized synthetic chromosome, and stable transformation of the chromosome into the engineered improved host strains for use in production of biofuels via biorefinery operations.

Lignocellulosic biomass is a sustainable feedstock for biorefineries, but inefficient xylose utilization limits microbial bioproduction. Here, the oleaginous yeast Yarrowia lipolytica was engineered to produce succinic acid (SA) from xylose by resolving metabolic and regulatory conflicts. Initial overexpression of xylose catabolic genes (XR, XDH, XK) in an SA‐hyperproducing strain did not activate xylose utilization, indicating underlying cryptic constraints. Adaptive evolution identified critical mutations (Snf1R78W, Scp1delGTC) that globally downregulated downstream pathways, including glycolysis and β‐oxidation, restoring growth using xylose but reducing SA production. To overcome this trade‐off, a random expression library strategy incorporating multi‐copy amplification of XR, XDH, and XK genes via nonhomologous end joining (NHEJ) was employed. This approach significantly enhanced xylose utilization and SA production, achieving 83.78 g L−1 SA from corn stover hydrolysate at pH 3.5 (yield: 0.66 g g−1 mixed sugars; productivity: 1.21 g L−1 h−1). Mechanistic studies revealed that fatty acid metabolism drives a futile cycle converting cytosolic NADPH to mitochondrial NADH, essential for SA biosynthesis via the reductive TCA pathway. This cycle competitively inhibits xylose catabolism unless pathway genes are amplified to balance cofactor demand. This work highlights the importance of fatty acid metabolism in Y. lipolytica for SA biosynthesis, cofactor rebalancing, and pathway cross‐talks.