Engineering the cellulolytic bacterium, Clostridium thermocellum, to co-utilize hemicellulose.

Consolidated bioprocessing (CBP) of lignocellulosic biomass holds promise to realize economic production of second-generation biofuels/chemicals, and Clostridium thermocellum is a leading candidate for CBP due to it being one of the fastest degraders of crystalline cellulose and lignocellulosic biomass. However, CBP by C. thermocellum is approached with co-cultures, because C. thermocellum does not utilize hemicellulose. When compared with a single-species fermentation, the co-culture system introduces unnecessary process complexity that may compromise process robustness. In this study, we engineered C. thermocellum to co-utilize hemicellulose without the need for co-culture. By evolving our previously engineered xylose-utilizing strain in xylose, an evolved clonal isolate (KJC19-9) was obtained and showed improved specific growth rate on xylose by ∼3-fold and displayed comparable growth to a minimally engineered strain grown on the bacteria's naturally preferred substrate, cellobiose. To enable full xylan deconstruction to xylose, we recombinantly expressed three different ß-xylosidase enzymes originating from Thermoanaerobacterium saccharolyticum into KJC19-9 and demonstrated growth on xylan with one of the enzymes. This recombinant strain was capable of co-utilizing cellulose and xylan simultaneously, and we integrated the ß-xylosidase gene into the KJC19-9 genome, creating the KJCBXint strain. The strain, KJC19-9, consumed monomeric xylose but accumulated xylobiose when grown on pretreated corn stover, whereas the final KJCBXint strain showed significantly greater deconstruction of xylan and xylobiose. This is the first reported C. thermocellum strain capable of degrading and assimilating hemicellulose polysaccharide while retaining its cellulolytic capabilities, unlocking significant potential for CBP in advancing the bioeconomy.

Engineering Saccharomyces cerevisiae for targeted hydrolysis and fermentation of glucuronoxylan through CRISPR/Cas9 genome editing.

BACKGROUND: The abundance of glucuronoxylan (GX) in agricultural and forestry residual side streams positions it as a promising feedstock for microbial conversion into valuable compounds. By engineering strains of the widely employed cell factory Saccharomyces cerevisiae with the ability to directly hydrolyze and ferment GX polymers, we can avoid the need for harsh chemical pretreatments and costly enzymatic hydrolysis steps prior to fermentation. However, for an economically viable bioproduction process, the engineered strains must efficiently express and secrete enzymes that act in synergy to hydrolyze the targeted polymers. RESULTS: The aim of this study was to equip the xylose-fermenting S. cerevisiae strain CEN.PK XXX with xylanolytic enzymes targeting beechwood GX. Using a targeted enzyme approach, we matched hydrolytic enzyme activities to the chemical features of the GX substrate and determined that besides endo-1,4-ß-xylanase and ß-xylosidase activities, α-methyl-glucuronidase activity was of great importance for GX hydrolysis and yeast growth. We also created a library of strains expressing different combinations of enzymes, and screened for yeast strains that could express and secrete the enzymes and metabolize the GX hydrolysis products efficiently. While strains engineered with BmXyn11A xylanase and XylA ß-xylosidase could grow relatively well in beechwood GX, strains further engineered with Agu115 α-methyl-glucuronidase did not display an additional growth benefit, likely due to inefficient expression and secretion of this enzyme. Co-cultures of strains expressing complementary enzymes as well as external enzyme supplementation boosted yeast growth and ethanol fermentation of GX, and ethanol titers reached a maximum of 1.33 g L- 1 after 48 h under oxygen limited condition in bioreactor fermentations. CONCLUSION: This work underscored the importance of identifying an optimal enzyme combination for successful engineering of S. cerevisiae strains that can hydrolyze and assimilate GX. The enzymes must exhibit high and balanced activities, be compatible with the yeast's expression and secretion system, and the nature of the hydrolysis products must be such that they can be taken up and metabolized by the yeast. The engineered strains, particularly when co-cultivated, display robust growth and fermentation of GX, and represent a significant step forward towards a sustainable and cost-effective bioprocessing of GX-rich biomass. They also provide valuable insights for future strain and process development targets.

Metabolic engineering of Caldicellulosiruptor bescii for hydrogen production.

Hydrogen is an alternative fuel for transportation vehicles because it is clean, sustainable, and highly flammable. However, the production of hydrogen from lignocellulosic biomass by microorganisms presents challenges. This microbial process involves multiple complex steps, including thermal, chemical, and mechanical treatment of biomass to remove hemicellulose and lignin, as well as enzymatic hydrolysis to solubilize the plant cell walls. These steps not only incur costs but also result in the production of toxic hydrolysates, which inhibit microbial growth. A hyper-thermophilic bacterium of Caldicellulosiruptor bescii can produce hydrogen by decomposing and fermenting plant biomass without the need for conventional pretreatment. It is considered as a consolidated bioprocessing (CBP) microorganism. This review summarizes the basic scientific knowledge and hydrogen-producing capacity of C. bescii. Its genetic system and metabolic engineering strategies to improve hydrogen production are also discussed. KEY POINTS: • Hydrogen is an alternative and eco-friendly fuel. • Caldicellulosiruptor bescii produces hydrogen with a high yield in nature. • Metabolic engineering can make C. bescii to improve hydrogen production.

Adaptive evolution of Clostridium thermocellum ATCC 27405 on alternate carbon sources leads to altered fermentation profiles.

Consolidated bioprocessing candidate, Clostridium thermocellum, is a cellulose hydrolysis specialist, with the ability to ferment the released sugars to produce bioethanol. C. thermocellum is generally studied with model substrates Avicel and cellobiose to understand the metabolic pathway leading to ethanol. In the present study, adaptive laboratory evolution, allowing C. thermocellum DSM 1237 to adapt to growth on glucose, fructose, and sorbitol, with the prospect that some strains will adapt their metabolism to yield more ethanol. Adaptive growth on glucose and sorbitol resulted in an approximately 1 mM and 2 mM increase in ethanol yield per millimolar glucose equivalent, respectively, accompanied by a shift in the production of the other expected fermentation end products. The increase in ethanol yield observed for sorbitol adapted cells was due to the carbon source being more reduced compared to cellobiose. Glucose and cellobiose have similar oxidation states thus the increase in ethanol yield is due to the rerouting of electrons from other reduced metabolic products excluding H2 which did not decrease in yield. There was no increase in ethanol yield observed for fructose adapted cells, but there was an unanticipated elimination of formate production, also observed in sorbitol adapted cells suggesting that fructose has regulatory implications on formate production either at the transcription or protein level.

Consolidated bioprocessing of lignocellulosic wastes in Northwest China for D-glucaric acid production by an artificial microbial consortium.

D-glucaric acid is a platform chemical of great importance and the consolidated bioprocessing (CBP) of lignocellulose by the microbial consortium of Trichoderma reesei C10 and Saccharomyces cerevisiae LGA-1C3S2 features prospects in biomanufacturing it. Here we compared some representative lignocelluloses in Northwest China including corn stover, wheat straw and switchgrass, and the leading pretreatments including steam explosion, subcritical water pretreatment, sodium hydroxide pretreatment, aqueous ammonia pretreatment, lime pretreatment, and diluted sulfuric acid pretreatment. It was found that sodium hydroxide pretreated switchgrass (SHPSG) was the best substrate for D-glucaric acid production, resulting in the highest D-glucaric acid titers, 11.69 ± 0.73 g/L in shake flask and 15.71 ± 0.80 g/L in 10L airlift fermenter, respectively. To the best of our knowledge, this is the highest D-glucaric acid production titer from lignocellulosic biomass. This work offers a paradigm of producing low-cost D-glucaric acid for low-carbon polyethylene 2,5-furandicarboxylate (PEF) and a reference on developing biorefinery in Northwest China.

Recent Advances in Food Waste Transformations into Essential Bioplastic Materials.

Lignocellulose is a major biopolymer in plant biomass with a complex structure and composition. It consists of a significant amount of high molecular aromatic compounds, particularly vanillin, syringeal, ferulic acid, and muconic acid, that could be converted into intracellular metabolites such as polyhydroxyalkanoates (PHA) and hydroxybutyrate (PHB), a key component of bioplastic production. Several pre-treatment methods were utilized to release monosaccharides, which are the precursors of the relevant pathway. The consolidated bioprocessing of lignocellulose-capable microbes for biomass depolymerization was discussed in this study. Carbon can be stored in a variety of forms, including PHAs, PHBs, wax esters, and triacylglycerides. From a biotechnology standpoint, these compounds are quite adaptable due to their precursors' utilization of hydrogen energy. This study lays the groundwork for the idea of lignocellulose valorization into value-added products through several significant dominant pathways.

Consolidated bioprocessing for bioethanol production by metabolically engineered cellulolytic fungus Myceliophthora thermophila.

Using cellulosic ethanol as fuel is one way to help achieve the world's decarbonization goals. However, the economics of the present technology are unfavorable, especially the cost of cellulose degradation. Here, we reprogram the thermophilic cellulosic fungus Myceliophthora thermophila to directly ferment cellulose into ethanol by mimicking the aerobic ethanol fermentation of yeast (the Crabtree effect), including optimizing the synthetic pathway, enhancing the glycolytic rate, inhibiting mitochondrial NADH shuttles, and knocking out ethanol consumption pathway. The final engineered strain produced 52.8 g/L ethanol directly from cellulose, and 39.8 g/L from corncob, without the need for any added cellulase, while the starting strain produced almost no ethanol. We also demonstrate that as the ethanol fermentation by engineered M. thermophila increases, the composition and expression of cellulases that facilitate the degradation of cellulose, especially cellobiohydrolases, changes. The simplified production process and significantly increased ethanol yield indicate that the fungal consolidated bioprocessing technology that we develop here (one-step, one-strain ethanol production) is promising for fueling sustainable carbon-neutral biomanufacturing in the future.

Effect of itaconic acid production on Neurospora crassa in consolidated bioprocessing of cellulose.

A system for itaconic acid synthesis from cellulose by Neurospora crassa was established, resulting in the highest yield of itaconic acid was 354.08 + 35.99 mg/L. Meanwhile, cellulase activity increased significantly, without any strain modifications for improved cellulase production. Multi-omics analyses showed that itaconic acid synthesis reduced energy production, leading to decreases in trehalose, cell wall, fatty acids synthesis and downregulations in MAPK signaling pathway, cell cycle and meiosis. More importantly, the low-energy environment enhanced the energy-efficient cellobionic acid/gluconic acid pathway, and the cellulase composition also changed significantly, manifested as the up-regulation of LPMOs and the down-regulation of ß-glucosidases. Enhancing LPMOs-cellobionic acid/gluconic acid system has the potential to reduce energy consumption of the consolidated bioprocessing. These findings offer an overview of resource allocations by N. crassa in response to itaconic acid synthesis and highlight a series of intriguing connections between itaconic acid synthesis and cellulase synthesis in consolidated bioprocessing.

Regulation of lignocellulose degradation in microorganisms.

Microbial strategies for biomass deconstruction involve an incredible repertoire of enzymatic, structural, and regulatory proteins. From carbohydrate active enzymes to cellulosomes, bacteria, yeast, and filamentous fungi adapt their functional machinery to grow from alternative carbon sources such as lignocellulose and survive starvation. In that context, microbes must be able to sense, bind, degrade, and utilize lignin, cellulose, and hemicelluloses. Nature has developed specialized protein modules, RNA structures, and regulatory systems operating at a genomic, transcription, and translation level. This review briefly summarizes the main regulatory pathways involved in lignocellulose microbial degradation, including carbon catabolite repression; anti-sigma factors; regulatory RNA elements such as small RNAs, antisense RNA, RNA-binding proteins, and selective RNA processing and stabilization; and transcriptional regulators and unfolded protein response. Interplay with global regulators controlling pH response and nitrogen utilization is also revised.

Engineering natural isolates of Saccharomyces cerevisiae for consolidated bioprocessing of cellulosic feedstocks.

Saccharomyces cerevisiae has gained much attention as a potential host for cellulosic bioethanol production using consolidated bioprocessing (CBP) methodologies, due to its high-ethanol-producing titres, heterologous protein production capabilities, and tolerance to various industry-relevant stresses. Since the secretion levels of heterologous proteins are generally low in domesticated strains of S. cerevisiae, natural isolates may offer a more diverse genetic background for improved heterologous protein secretion, while also displaying greater robustness to process stresses. In this study, the potential of natural and industrial S. cerevisiae strains to secrete a core set of cellulases (CBH1, CBH2, EG2, and BGL1), encoded by genes integrated using CRISPR/Cas9 tools, was evaluated. High levels of heterologous protein production were associated with a reduced maximal growth rate and with slight changes in overall strain robustness, compared to the parental strains. The natural isolate derivatives YI13_BECC and YI59_BECC displayed superior secretion capacity for the heterologous cellulases at high incubation temperature and in the presence of acetic acid, respectively, compared to the reference industrial strain MH1000_BECC. These strains also exhibited multi-tolerance to several fermentation-associated and secretion stresses. Cultivation of the strains on crystalline cellulose in oxygen-limited conditions yielded ethanol concentrations in the range of 4-4.5 g/L, representing 35-40% of the theoretical maximum ethanol yield after 120 h, without the addition of exogenous enzymes. This study therefore highlights the potential of these natural isolates to be used as chassis organisms in CBP bioethanol production. KEY POINTS: • Process-related fermentation stresses influence heterologous protein production. • Transformants produced up to 4.5 g/L ethanol, ~ 40% of the theoretical yield in CBP. • CRISPR/Cas9 was feasible for integrating genes in natural S. cerevisiae isolates.

Metabolic engineering of Aspergillus niger to enhance production of ethanol.

This work describes the genetic transformation of a strain of Aspergillus niger with five different constructs containing 16 different heterologous genes, coding for four oxidoreductases, two cellobiohydrolases, one endoglucanase, one ß-glucosidase, six enzymes involved in xylose metabolism, and two enzymes involved in fermentation. The aim was to try and engineer a consolidated bioprocessing in A. niger. The fungus already contains most of these enzymes and we only enhanced endogenous activities. We recovered nine transformants containing all genes, as indicated by polymerase chain reaction (PCR). To confirm that the products of the genes were functional, we measured the activity of five different enzymes in all the strains, and they all showed enhanced activity over the wild-type (wt) strain. The strains were grown on carboxymethyl cellulose (CMC) and xylan as substrates, and they produced considerably more ethanol than the wt. The levels of ethanol production were comparable to those reported in the literature.

Enhancing Secretion of Endoglucanase in Zymomonas mobilis by Disturbing Peptidoglycan Synthesis.

Zymomonas mobilis (Z. mobilis) is a potential candidate strain for consolidated bioprocessing (CBP) in lignocellulosic biorefinery. However, the low-level secretion of cellulases limits this CBP process, and the mechanism of protein secretion that is affected by cell wall peptidoglycan is also not well understood. Here, we constructed several penicillin-binding protein (PBP)-deficient strains derived from Z. mobilis S192 to perturb the cell wall peptidoglycan network and then investigated the effects of peptidoglycan on the endoglucanase secretion. The results showed that extracellular recombinant endoglucanase production was significantly enhanced in PBP mutant strains, notably, Δ1089/0959 (4.09-fold) and Δ0959 (5.76-fold) in comparison to parent strains. For PBP-deficient strains, the growth performance was not significantly inhibited, but cell morphology was altered. In addition, enhanced antibiotic sensitivity and reduced inhibitor tolerance were also detected in our study. The concentration of intracellular soluble peptidoglycan was increased, especially for single-gene deletion. Outer membrane permeability of PBP-deficient strains was also improved, notably, Δ1089/0959 (1.14-fold) and Δ0959 (1.07-fold), which might explain the increased endoglucanase extracellular secretion. Our findings indicated that PBP-deficient Z. mobilis was capable of increasing endoglucanase extracellular secretion via cell wall peptidoglycan disturbance, and it will provide a foundation for the development of CBP technology in Z. mobilis in the future. IMPORTANCE Cell wall peptidoglycan has the function to maintain cell robustness and acts as the barrier to secret recombinant proteins from the cytoplasm to extracellular space in Z. mobilis and other bacteria. Herein, we perturbed the peptidoglycan synthesis network via knocking out PBPs (ZMO0197, ZMO0959, ZMO1089) to enhance recombinant endoglycanase extracellular secretion in Z. mobilis S912. This study could lay the foundation for understanding the regulatory network of cell wall synthesis and guide the construction of CBP strains in Z. mobilis.

Improvement of cell-tethered cellulase activity in recombinant strains of Saccharomyces cerevisiae.

Consolidated bioprocessing (CBP) remains an attractive option for the production of commodity products from pretreated lignocellulose if a process-suitable organism can be engineered. The yeast Saccharomyces cerevisiae requires engineered cellulolytic activity to enable its use in CBP production of second-generation (2G) bioethanol. A promising strategy for heterologous cellulase production in yeast entails displaying enzymes on the cell surface by means of glycosylphosphatidylinositol (GPI) anchors. While strains producing a core set of cell-adhered cellulases that enabled crystalline cellulose hydrolysis have been created, secreted levels of enzyme were insufficient for complete cellulose hydrolysis. In fact, all reported recombinant yeast CBP candidates must overcome the drawback of generally low secretion titers. Rational strain engineering can be applied to enhance the secretion phenotype. This study aimed to improve the amount of cell-adhered cellulase activities of recombinant S. cerevisiae strains expressing a core set of four cellulases, through overexpression of genes that were previously shown to enhance cellulase secretion. Results showed significant increases in cellulolytic activity for all cell-adhered cellulase enzyme types. Cell-adhered cellobiohydrolase activity was improved by up to 101%, ß-glucosidase activity by up to 99%, and endoglucanase activity by up to 231%. Improved hydrolysis of crystalline cellulose of up to 186% and improved ethanol yields from this substrate of 40-50% in different strain backgrounds were also observed. In addition, improvement in resistance to fermentation stressors was noted in some strains. These strains represent a step towards more efficient organisms for use in 2G biofuel production. KEY POINTS: • Cell-surface-adhered cellulase activity was improved in strains engineered for CBP. • Levels of improvement of activity were strain and enzyme dependent. • Crystalline cellulose conversion to ethanol could be improved up to 50%.

Coexpression of a ß-d-Xylosidase from Thermotoga maritima and a Family 10 Xylanase from Acidothermus cellulolyticus Significantly Improves the Xylan Degradation Activity of the Caldicellulosiruptor bescii Exoproteome.

Caldicellulosiruptor species are hyperthermophilic, Gram-positive anaerobes and the most thermophilic cellulolytic bacteria so far described. They have been engineered to convert switchgrass to ethanol without pretreatment and represent a promising platform for the production of fuels, chemicals, and materials from plant biomass. Xylooligomers, such as xylobiose and xylotriose, that result from the breakdown of plant biomass more strongly inhibit cellulase activity than do glucose or cellobiose. High concentrations of xylobiose and xylotriose are present in C. bescii fermentations after 90 h of incubation, and removal or breakdown of these types of xylooligomers is crucial to achieving high conversion of plant biomass to product. In previous studies, the addition of exogenous ß-d-xylosidase substantially improved the performance of glucanases and xylanases in vitro. ß-d-Xylosidases are, in fact, essential enzymes in commercial preparations for efficient deconstruction of plant biomass. In addition, the combination of xylanase and ß-d-xylosidase is known to exhibit synergistic action on xylan degradation. In spite of its ability to grow efficiently on xylan substrates, no extracellular ß-d-xylosidase was identified in the C. bescii genome. Here, we report that the coexpression of a thermal stable ß-d-xylosidase from Thermotoga maritima and a xylanase from Acidothermus cellulolyticus in a C. bescii strain containing the A. cellulolyticus E1 endoglucanase significantly increased the activity of the exoproteome as well as growth on xylan substrates. The combination of these enzymes also resulted in increased growth on crystalline cellulose in the presence of exogenous xylan. IMPORTANCECaldicellulosiruptor species are bacteria that grow at extremely high temperature, more than 75°C, and are the most thermophilic bacteria so far described that are capable of growth on plant biomass. This native ability allows the use of unpretreated biomass as a growth substrate, eliminating the prohibitive cost of preprocessing/pretreatment of the biomass. They only grow under strictly anaerobic conditions, and the combination of high temperature and the lack of oxygen reduces the cost of fermentation and contamination by other microbes. They have been genetically engineered to convert switchgrass to ethanol without pretreatment and represent a promising platform for the production of fuels, chemicals, and materials from plant biomass. In this study, we introduced genes from other cellulolytic bacteria and identified a combination of enzymes that improves growth on plant biomass. An important feature of this study is that it measures growth, validating predictions made from adding enzyme mixtures to biomass.

Modeling coordinated enzymatic control of saccharification and fermentation by Clostridium thermocellum during consolidated bioprocessing of cellulose.

Consolidated bioprocessing (CBP) of cellulose is a cost-effective route to produce valuable biochemicals by integrating saccharification, fermentation and cellulase synthesis in a single step. However, the lack of understanding of governing factors of interdependent saccharification and fermentation in CBP eludes reliable process optimization. Here, we propose a new framework that synergistically couples population balances (to simulate cellulose depolymerization) and cybernetic models (to model enzymatic regulation of fermentation) to enable improved understanding of CBP. The resulting framework, named the unified cybernetic-population balance model (UC-PBM), enables simulation of CBP driven by coordinated control of enzyme synthesis through closed-loop interactions. UC-PBM considers two key aspects in controlling CBP: (1) heterogeneity in cellulose properties and (2) cellular regulation of competing cell growth and cellulase secretion. In a case study on Clostridium thermocellum, UC-PBM not only provides a decent fit with various exometabolomic data, but also reveals that: (i) growth-decoupled cellulase-secreting pathways are only activated during famine conditions to promote the production of growth substrates, and (ii) starting cellulose concentration has a strong influence on the overall flux distribution. Equipped with mechanisms of cellulose degradation and fermentative regulations, UC-PBM is practical to explore phenotypic functions for primary evaluation of microorganisms' potential for metabolic engineering and optimal design of bioprocess.

Microbial application of thermophilic Thermoanaerobacterium species in lignocellulosic biorefinery.

Recently, thermophilic Thermoanaerobacterium species have attracted increasing attentions in consolidated bioprocessing (CBP), which can directly utilize lignocellulosic materials for biofuels production. Compared to the mesophilic process, thermophilic process shows greater prospects in CBP due to its relatively highly efficiency of lignocellulose degradation. In addition, thermophilic conditions can avoid microbial contamination, reduce the cooling costs, and further facilitate the downstream product recovery. However, only few reviews specifically focused on the microbial applications of thermophilic Thermoanaerobacterium species in lignocellulosic biorefinery. Accordingly, this review will comprehensively summarize the recent advances of Thermoanaerobacterium species in lignocellulosic biorefinery, including their secreted xylanases and bioenergy production. Furthermore, the co-culture can significantly reduce the metabolic burden and achieve the more complex work, which will be discussed as the further perspectives. KEY POINTS: • Thermoanaerobacterium species, promising chassis for lignocellulosic biorefinery. • Potential capability of hemicellulose degradation for Thermoanaerobacterium species. • Efficient bioenergy production by Thermoanaerobacterium species through metabolic engineering.

Stress modulation as a means to improve yeasts for lignocellulose bioconversion.

The second-generation (2G) fermentation environment for lignocellulose conversion presents unique challenges to the fermentative organism that do not necessarily exist in other industrial fermentations. While extreme osmotic, heat, and nutrient starvation stresses are observed in sugar- and starch-based fermentation environments, additional pre-treatment-derived inhibitor stress, potentially exacerbated by stresses such as pH and product tolerance, exist in the 2G environment. Furthermore, in a consolidated bioprocessing (CBP) context, the organism is also challenged to secrete enzymes that may themselves lead to unfolded protein response and other stresses. This review will discuss responses of the yeast Saccharomyces cerevisiae to 2G-specific stresses and stress modulation strategies that can be followed to improve yeasts for this application. We also explore published -omics data and discuss relevant rational engineering, reverse engineering, and adaptation strategies, with the view of identifying genes or alleles that will make positive contributions to the overall robustness of 2G industrial strains. KEYPOINTS: • Stress tolerance is a key driver to successful application of yeast strains in biorefineries. • A wealth of data regarding stress responses has been gained through omics studies. • Integration of this knowledge could inform engineering of fit for purpose strains.

Cross-feeding and wheat straw extractives enhance growth of Clostridium thermocellum-containing co-cultures for consolidated bioprocessing.

Co-cultures consisting of three thermophilic and lignocellulolytic bacteria, namely Clostridium thermocellum, C. stercorarium, and Thermoanaerobacter thermohydrosulfuricus, degrade lignocellulosic material in a synergistic manner. When cultured in a defined minimal medium two of the members appeared to be auxotrophic and unable to grow, but the growth of all species was observed in all co-culture combinations, indicating cross-feeding of unidentified growth factors between the members. Growth factors also appeared to be present in water-soluble extractives obtained from wheat straw, allowing for the growth of the auxotrophic monocultures in the defined minimal medium. Cell enumeration during growth on wheat straw in this medium revealed different growth profiles of the members that varied between the co-cultures. End-product profiles also varied substantially between the cultures, with significantly higher ethanol production in all co-cultures compared to the mono-cultures. Understanding interactions between co-culture members, and the additional nutrients provided by lignocellulosic substrates, will aid us in consolidated bioprocessing design.

Dynamic consolidated bioprocessing for direct production of xylonate and shikimate from xylan by Escherichia coli.

Numerous value-added chemicals can be produced using xylan as a feedstock. However, the product yields are limited by low xylan utilization efficiency, as well as by carbon flux competition between biomass production and biosynthesis. Herein, a dynamic consolidated bioprocessing strategy was developed, which coupled xylan utilization and yield optimization modules. Specifically, we achieved the efficient conversion of xylan to valuable chemicals in a fully consolidated manner by optimizing the expression level of xylanases and xylose transporter in the xylan utilization module. Moreover, a cell density-dependent, and Cre-triggered dynamic system that enabled the dynamic decoupling of biosynthesis and biomass production was constructed in the yield optimization module. The final shake flask-scale titers of xylonate, produced through an exogenous pathway, and shikimate, produced through an endogenous pathway, reached 16.85 and 3.2 g L-1, respectively. This study not only provides an efficient microbial platform for the utilization of xylan, but also opens up the possibility for the large-scale production of high value-added chemicals from renewable feedstocks.

Simultaneous secretion of seven lignocellulolytic enzymes by an industrial second-generation yeast strain enables efficient ethanol production from multiple polymeric substrates.

A major hurdle in the production of bioethanol with second-generation feedstocks is the high cost of the enzymes for saccharification of the lignocellulosic biomass into fermentable sugars. Simultaneous saccharification and fermentation with Saccharomyces cerevisiae yeast that secretes a range of lignocellulolytic enzymes might address this problem, ideally leading to consolidated bioprocessing. However, it has been unclear how many enzymes can be secreted simultaneously and what the consequences would be on the C6 and C5 sugar fermentation performance and robustness of the second-generation yeast strain. We have successfully expressed seven secreted lignocellulolytic enzymes, namely endoglucanase, ß-glucosidase, cellobiohydrolase I and II, xylanase, ß-xylosidase and acetylxylan esterase, in a single second-generation industrial S. cerevisiae strain, reaching 94.5 FPU/g CDW and enabling direct conversion of lignocellulosic substrates into ethanol without preceding enzyme treatment. Neither glucose nor the engineered xylose fermentation were significantly affected by the heterologous enzyme secretion. This strain can therefore serve as a promising industrial platform strain for development of yeast cell factories that can significantly reduce the enzyme cost for saccharification of lignocellulosic feedstocks.